GENES
The present invention relates to two primordial germ cell-specific expressed genes, Fragilis and Stella. The sequences and sues of human Stella and Fragilis are disclosed herein, as are several mouse sequences related to Fragilis. The present invention relates to the use of Stella and Fragilis as markers for primordial germ cells and can be used to identify such cells. Additionally, the present invention relates to the use of Stella and Fragilis for the diagnosis, treatment and/or prevention of disease.
The present invention relates to the fields of development, molecular biology and genetics. More particularly, the invention relates to genes which are expressed exclusively in the earliest populations of primordial germ cells (PGCs) and the use of such genes and the products thereof in identification of pluripotent and multipotent cells such as PGCs, pluripotent embryonic stem cells (ES) and pluripotent embryonic germ cells (EG), in cell populations. They are also markers for a change in the state of cells from being non pluripotent to becoming pluripotent, and in being able to confer this state on a non pluripotent cell.
INTRODUCTIONPost fertilisation, the early mammalian embryo undergoes four rounds of cleavage to form a morula of 16 cells. These cells, following further rounds of division, develop into a blastocyst in which the cells can be divided into two distinct regions; the inner cell mass, which will form the embryo, and the trophectoderm, which will form extra-embryonic tissue, such as the placenta.
The cells that form part of the embryo up until the formation of the blastocyst are totipotent; in other words, each of the cells has the ability to give rise to a complete individual embryo, and to all the extra-embryonic tissues required for its development. After blastocyst formation, the cells of the inner cell mass are no longer totipotent, but are pluripotent, in that they can give rise to a range of different tissues. A known marker for such cells is the expression of the enzyme alkaline phosphatase and Oct4.
Primordial germ cells (PGCs) are pluripotent cells that have the ability to differentiate into all three primary germ layers. In mammals, the PGCs migrate from the base of the allantois, through the hindgut epithelium and dorsal mesentery, to colonise the gonadal anlague. The PGC-derived cells have a characteristically low cytoplasm/nucleus ratio, usually with prominent nucleoli. PGCs may be isolated from the embryos by removing the genital ridge of the embryo, dissociating the PGCs from the gonadal anlague, and collecting the PGCs. The earliest PGC population is reported to consist of a cluster of some 45 (forty-five) alkaline phosphatase positive cells, found at the base of the emerging allantois, 7.25 days post-fertilisation (Ginsburg et al., (1990) Development 110:521-528).
PGCs have many applications in modern biotechnology and molecular biology. They are useful in the production of transgenic animals, where embryonic germ (EG) cells derived from PGCs may be used in much the same manner as embryonic stem (ES) cells (Labosky et al., (1994) Development 120:3197-3204). Moreover, they are useful in the study of foetal development and the provision of pluripotent stem cells for tissue regeneration in the therapy of degenerative diseases and repopulation of damaged tissue following trauma. Above all, PGCs while having some specialised properties, retain an underlying pluripotency, which is lost from the neighbouring cells that surround the founder population of PGCs that acquire a somatic cell fate. PGCs and the surrounding somatic cells share a common ancestry. However, the founder PGCs are few in number and difficult to isolate from embryonic tissue and the surrounding somatic cells, which complicates their study and the development of techniques which make use thereof.
Little is known in the art about the expression of genes in the founder population of PGCs and the relationship between PGC-specific gene expression and the retention of pluripotency in these cells. Certain markers for PGCs are known—for example, the expression of tissue non-specific alkaline phosphatase (TNAP) has been used as a marker for early PGCs (Ginsburg et al., (1990) Development 110:521-528). Oct4 is known to be expressed in PGCs, but not somatic cells (Yoem et al., (1996) Development 122:881-894). Other markers, such as BMP4, are known to be expressed primarily in somatic tissues (Lawson et al., (1999) Genes & Dev. 13:424-436). However, none of these genes is specific for PGCs, since they are also expressed in other tissue types. There is therefore a need in the art for the identification of genes which may be used as markers for PGCs and which may provide an insight into the biology of germ cell development and the nature of the pluripotent state.
Our unpublished International Patent Application Number PCT/GB02/00215, the contents of which are hereby incorporated by reference, discloses sequences and uses of two rodent genes, Stella and Fragilis. That document shows that Stella and Fragilis are expressed by PGCs and other pluripotent cells. The sequences of mouse Stella and Fragilis are also set out here as SEQ ID NO: 2 and SEQ ID NO: 1 respectively.
SUMMARYWe now disclose human sequences of Stella and Fragilis, as well as sequences of novel genes Fragilis 9-27, Fragilis 2, Fragilis 3, Fragilis 4, Fragilis 5 and Fragilis 6 comprising members of the Fragilis family of genes. We also disclose sequences of human Fragilis homologues, Ifitm 1, Ifitm 2, Ifitm 3 and Human ENSG142056. The genes and polypeptides described here, including associated products such as antibodies, are useful in a number of ways, for example, for diagnosis, treatment and/or prevention of diseases such as cancer.
According to a first aspect of the present invention, we provide a polypeptide comprising a human Stella amino acid sequence, preferably a sequence as shown in SEQ ID NO: 4, or a fragment, homologue, variant or derivative thereof.
There is provided, according to a second aspect of the present invention, a polypeptide comprising a human Fragilis amino acid sequence, preferably a sequence as shown in SEQ ID NO: 6, or a fragment, homologue, variant or derivative thereof.
We provide, according to a third aspect of the present invention, a polypeptide comprising an amino acid sequence selected from the group consisting of: Fragilis 9-27 (preferably comprising a sequence as shown in SEQ ID NO: 8), Fragilis 2 (preferably comprising a sequence as shown in SEQ ID NO: 10), Fragilis 3 (preferably comprising a sequence as shown in SEQ ID NO: 12), Fragilis 4 (preferably comprising a sequence as shown in SEQ ID NO: 14), Fragilis 5 (preferably comprising a sequence as shown in SEQ ID NO: 16) and Fragilis 6 (preferably comprising a sequence as shown in SEQ ID NO: 18), or a fragment, homologue, variant or derivative thereof.
As a fourth aspect of the present invention, there is provided a polypeptide consisting essentially of a sequence selected from the group consisting of: (a) the sequence of human Stella shown in SEQ ID NO: 4; (b) the sequence of human Fragilis shown in SEQ ID NO: 6; (c) the sequence of human Fragilis 9-27 shown in SEQ ID NO: 8; (d) the sequence of mouse Fragilis 2 shown in SEQ ID NO: 10; (e) the sequence of mouse Fragilis 3 shown in SEQ ID NO: 12; (f) the sequence of mouse Fragilis 4 shown in SEQ ID NO: 14; (g) the sequence of mouse Fragilis 5 shown in SEQ ID NO: 16; and (h) the sequence of mouse Fragilis 6 shown in SEQ ID NO: 18.
We provide, according to a fifth aspect of the present invention, a polypeptide which has at least 50%, 60%, 70%, 80%, 90% or 95% homology to a polypeptide according to any of the preceding aspects.
The present invention, in a sixth aspect, provides a polypeptide comprising a sequence of 15 or fewer contiguous amino acids, preferably a sequence of fewer than 10 contiguous amino acids, of a nucleic acid according to any of the preceding aspects.
In a seventh aspect of the present invention, there is provided a nucleic acid encoding a polypeptide according to any preceding aspect.
According to an eighth aspect of the present invention, we provide a nucleic acid sequence comprising a human Stella nucleic acid sequence, preferably a sequence as shown in SEQ ID NO: 3, or a fragment, homologue, variant or derivative thereof.
We provide, according to a ninth aspect of the invention, a nucleic acid sequence comprising a human Fragilis nucleic acid sequence, preferably a sequence as shown in SEQ ID NO: 5, or a fragment, homologue, variant or derivative thereof.
There is provided, in accordance with a tenth aspect of the present invention, a nucleic acid sequence comprising an Fragilis nucleic acid sequence acid sequence selected from the group consisting of: Fragilis 9-27 (preferably a sequence as shown in SEQ ID NO: 7), Fragilis 2 (preferably a sequence as shown in SEQ ID NO: 9), Fragilis 3 (preferably a sequence as shown in SEQ ID NO: 11), Fragilis 4 (preferably a sequence as shown in SEQ ID NO: 13), Fragilis 5 (preferably a sequence as shown in SEQ ID NO: 15) and Fragilis 6 (preferably a sequence as shown in SEQ ID NO: 17), or a fragment, homologue, variant or derivative thereof.
As an eleventh aspect of the invention, we provide a nucleic acid sequence consisting essentially of a sequence selected from the group consisting of: (a) the sequence of human Stella shown in SEQ ID NO: 3; (b) the sequence of human Fragilis shown in SEQ ID NO: 5; (c) the sequence of human Fragilis 9-27 shown in SEQ ID NO: 7; (d) the sequence of mouse Fragilis 2 shown in SEQ ID NO: 9; (e) the sequence of mouse Fragilis 3 shown in SEQ ID NO: 11; (f) the sequence of mouse Fragilis 4 shown in SEQ ID NO: 13; (g) the sequence of mouse Fragilis 5 shown in SEQ ID NO: 15; and (h) the sequence of mouse Fragilis 6 shown in SEQ ID NO: 17.
We provide, according to a twelfth aspect of the invention, there is provided a nucleic acid sequence which has at least 50%, 60%, 70%, 80%, 90% or 95% homology to a nucleic acid sequence as disclosed.
According to a thirteenth aspect of the present invention, we provide a nucleic acid comprising a sequence of 25 or fewer contiguous nucleotides, preferably a sequence of 15 contiguous nucleotides, of a nucleic acid as disclosed.
There is provided, according to a fourteenth aspect of the present invention, a complement of a nucleic acid sequence as disclosed.
We provide, according to a fifteenth aspect of the present invention, a nucleic acid comprising a nucleic acid sequence as disclosed, together with one or more nucleotide substitutions, in which the one or more substitutions do not alter the coding specificity of said nucleic acid as a result of the degeneracy of the genetic code.
According to a sixteenth aspect of the present invention, we provide a vector comprising such a nucleic acid sequence.
According to a seventeenth aspect of the present invention, we provide a host cell comprising a nucleic acid sequence as disclosed.
There is provided, according to a eighteenth aspect of the present invention, a method of producing a polypeptide as disclosed, the method comprising providing a nucleic acid as disclosed, a vector as disclosed, or a host cell as disclosed, and enabling the expression of a nucleic acid encoding the polypeptide.
We provide, according to a nineteenth aspect of the invention, a method for identifying a pluripotent cell, comprising detecting such a polypeptide, or such a nucleic acid in the cell.
Preferably, the method comprises the steps of amplifying nucleic acids from a putative pluripotent cell using 5′ and 3′ primers specific for Stella and/or Fragilis, and detecting amplified nucleic acid thus produced. Preferably, the nucleic acid sequence is detected by in situ hybridisation. Preferably, the nucleic acid sequence is detected by detecting a protein product encoded by the nucleic acid. Preferably, the polypeptide is detected by immunostaining.
There is provided, according to a twentieth aspect of the present invention, an antibody specific for a polypeptide as disclosed. Preferably, the antibody is capable of specifically binding to an extracellular domain of Fragilis.
We provide, according to a twenty-first aspect of the present invention, use of such an antibody for the identification and/or isolation of a pluripotent cell.
As a twenty-second aspect of the present invention, there is provided pluripotent cell identified by a method as disclosed.
We provide, according to a twenty-third aspect of the present invention, a method of treatment or prophylaxis of a disease in an individual, the method comprising modulating the expression and/or the activity of Stella and/or Fragilis in a cell of the individual.
Preferably, the method comprises up-regulating the expression and/or the activity of Stella and/or Fragilis.
The present invention, in a twenty-fourth aspect, provides a Stella or Fragilis polypeptide or nucleic acid, or an antibody against Stella or Fragilis, for use in a method of treatment or prophylaxis of a disease in an individual.
In a twenty-fifth aspect of the present invention, there is provided use of a Stella or Fragilis polypeptide or nucleic acid, or an antibody against Stella or Fragilis, for the preparation of a pharmaceutical composition for the treatment of a disease.
According to an twenty-sixth aspect of the present invention, we provide a method of diagnosis of a disease, the method comprising detecting a modulation of expression and/or activity of Stella and/or Fragilis in a cell of an individual.
In preferred embodiments, the disease is selected from the group consisting of: testis tumor, colon tumor, stomach, germ cell tumors, choriocarcinoma, lung, large cell carcinoma, uterus, and leiomyosarcoma Stella is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
We provide, according to a twenty-seventh aspect of the invention, a method of identifying a molecule capable of binding to Stella or Fragilis, the method comprising the steps of: (a) providing a Stella or Fragilis polypeptide; (b) contacting the Stella or Fragilis polypeptide with a candidate molecule; and (c) detecting binding between the candidate molecule and the Stella or Fragilis polypeptide, as the case may be.
There is provided, according to a twenty-eighth aspect of the present invention, a method of identifying an agonist or antagonist of Stella or Fragilis, the method comprising the steps of: (a) providing a Stella or Fragilis polypeptide; (b) contacting the Stella or Fragilis polypeptide with a candidate molecule; and (c) detecting modulation of an activity of the Stella or Fragilis polypeptide.
Preferably, the polypeptide is contacted with a plurality of candidate molecules, preferably in the form of a library.
We provide, according to a twenty-ninth aspect of the present invention, use of an antagonist of Stella or Fragilis, preferably as identified by a method according to any of the previous aspects, in a method of treatment or prophylaxis of a disease in an individual.
SEQ ID NO: 1 shows the nucleic acid sequence of mouse Fragilis while SEQ ID NO: 2 shows the nucleic acid sequence of mouse Stella.
SEQ ID NO: 3 shows the nucleic acid sequence of human Stella; SEQ ID NO: 4 shows the amino acid sequence of human Stella; SEQ ID NO: 5 shows the nucleic acid sequence of human Fragilis 1-8D; SEQ ID NO: 6 shows the amino acid sequence of human Fragilis 1-8D; SEQ ID NO: 7 shows the nucleic acid sequence of human Fragilis 9-27/Leu13; SEQ ID NO: 8 shows the amino acid sequence of human Fragilis 9-27/Leu13; SEQ ID NO: 9 shows the nucleic acid sequence of mouse Fragilis 2; SEQ ID NO: 10 shows the amino acid sequence of mouse Fragilis 2; SEQ ID NO: 11 shows the nucleic acid sequence of mouse Fragilis 3; SEQ ID NO: 12 shows the amino acid sequence of mouse Fragilis 3; SEQ ID NO: 13 shows the nucleic acid sequence of mouse Fragilis 4; SEQ ID NO: 14 shows the amino acid sequence of mouse Fragilis 4; SEQ ID NO: 15 shows the nucleic acid sequence of mouse Fragilis 5; SEQ ID NO: 16 shows the amino acid sequence of mouse Fragilis 5; SEQ ID NO: 17 shows the nucleic acid sequence of mouse Fragilis 6; SEQ ID NO: 18 shows the amino acid sequence of mouse Fragilis 6.
DETAILED DESCRIPTIONThe practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-3142), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is herein incorporated by reference.
Stella and FragilisThe disclosure provides generally for Fragilis and Stella nucleic acids, polypeptides, as well as fragments, homologues, variants and derivatives thereof. In particular, we provide for human Stella and Fragilis nucleic acids, polypeptides, as well as fragments, etc.
The names “Fragilis” and “GCR1” should be understood as synonymous with each other, and likewise, “Stella” and “GCR2” should be considered synonyms. In general, unless the context requires otherwise, the term “Fragilis” should be taken to refer to any member of the Fragilis family, including Fragilis itself, Fragilis 9-27, Fragilis 2, Fragilis 3, Fragilis 4, Fragilis 5 and Fragilis 6. In addition, Ifitm1, Ifitm2, Ifitm3 and ENSG142056 are preferably included within the term “Fragilis”. Preferably, the Stella and Fragilis sequences disclosed are derived from higher animals, for example primates, in particular, Homo sapiens (man).
In preferred embodiments, therefore, Fragilis should be taken to refer to the human nucleic acid sequence shown in SEQ ID NO: 5, or the human amino acid sequence shown in SEQ ID NO: 6, as the context requires. Furthermore, in preferred embodiments, Stella should be taken to refer to the human nucleic acid sequence shown in SEQ ID NO: 3, or the human amino acid sequence shown in SEQ ID NO: 4, as the context requires.
Human Stella and Fragilis are related by homology and function to the mouse Stella and Fragilis sequences disclosed in PCT/GB02/00215, and also set out as SEQ ID NO: 1 and SEQ ID NO: 2 in this document. Fragilis and Stella are PGC-specific transcripts. Fragilis is upregulated during the process of lineage commitment of PGCs, while Stella is upregulated after Fragilis, and marks commitment to the PGC fate.
Human Fragilis (also known as Germ cell restricted-1, GCR1), encodes a 132 amino acid protein with a predicted molecular weight of 14.5 kD. A best fit model of the EMBL program PredictProtein predicts two transmembrane domains, both N and C terminus ends being located outside. A BLASTP search revealed that Fragilis is a novel member of the interferon-inducible protein family. One prototype member, human 9-27 (identical to Leu-13 antigen), is inducible by interferon in leukocytes and endothelial cells, and is located at the cell surface as a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals (Deblandre, 1995). The BLASTN search revealed that the Fragilis sequence was found in ESTs derived from many different tissues both from embryos and adults, indicating that Fragilis may play a common role in different developmental and cell biological contexts, including cancer; this is described in further detail below. Database searches reveal a sequence match with the rat interferon-inducible protein (sp:INIB RAT, pir:JC1241) with unknown function. The Fragilis sequence appears several times in our screen, indicating high level expression in PGCs.
The second gene, human Stella, (Stella) encodes a 159 amino acid protein, of 17.5 kD. It has no sequence homology with any known protein, apart from murine and rodent Stella, contains several nuclear localisation consensus sequences and is highly basic pI, indicating a possible affinity to DNA. Furthermore a potential nuclear export signal is identified, indicating that Stella may shuttle between the nucleus and the cytoplasm. BLASTN analysis reveals that the Stella sequence is found only in the preimplantation embryo and germ line (newborn ovary, female 12.5 mesonephros and gonad etc.) ESTs indicating its predominant expression in totipotent and pluripotent cells. Interestingly, we found that Stella contains in its N terminus a modular domain which has some sequence similarity with the SAP motif. This motif is a putative DNA-binding domain involved in chromosomal organisation. Furthermore, the SMART program reveals the presence of a splicing factor motif-like structure in its C-terminus, These findings indicate a possible involvement of Stella in chromosomal organisation and RNA processing.
Antibodies may be raised against the Fragilis and/or Stella polypeptides. In particular, antibodies may be raised against the extracellular domain of Fragilis, which is a transmembrane polypeptide.
Antibodies, polypeptides and nucleic acids disclosed here are useful for the identification of PGCs in cell populations. The methods and compositions described here therefore provide a means to isolate PGCs, useful for example for the study of germ tissue development and the generation of transgenic animals, and PGCs when isolated by a method described here.
Homologues of Fragilis and Stella may also be used to identify PGCs and other pluripotent cells, such as ES or EG cells.
Furthermore, the methods and compositions described here are useful for treating or preventing cancer, in view of the role of Stella and Fragilis in development and cell fate control.
Human Stella and FragilisThe murine Stella and Fragilis genes are isolated by the methods shown in the Examples; these are disclosed in detail in our International Application PCT/GB02/00215. The murine Fragilis sequence isolated is shown as SEQ ID NO: 1, while the mouse Stella sequence is shown as SEQ ID NO: 2.
Human homologues of Stella and Fragilis are described in further detail in the following sections, and may (if necessary) be identified and cloned by standard techniques.
Human StellaHuman Stella Nucleic Acid Sequence
Human Stella Amino Acid Sequence
Human Stella Expression
Human Stella is expressed in a number of tissues, including: testis, colon, lung, uterus, and germinal center B cell.
Human Stella Diseases
Diseases associated with abnormal expression of human Stella include: testis tumor, colon tumor, stomach, germ cell tumors, choriocarcinoma, lung, large cell carcinoma, uterus, and leiomyosarcoma.
Human Stella is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Human FragilisHuman Fragilis is also referred to as Fragilis 1-8D.
Human Fragilis Nucleic Acid Sequence
Human Fragilis Amino Acid Sequence
Human Fragilis Genomic Location
Human Fragilis is located on chromosome 11, at chromosomal location 11p15.5.
Human Fragilis Diseases
Human Fragilis 1-8D is expressed in a number of tissues, including: stomach; adenocarcinoma; adenocarcinoma cell line; adipose; adrenal cortico adenoma for cushing's syndrome; adrenal gland; aorta; ascites; bone; bone marrow; brain; breast; breast_normal; cartilage; cervical carcinoma cell line; colon; colon_ins; colonic mucosa from 3 patients with crohn's disease; cord blood; corresponding non cancerous liver tissue; embryonal carcinoma; embryonal carcinoma, cell line; endometrium, adenocarcinoma cell line; epitheloid carcinoma; epitheloid carcinoma cell line; eye; foreskin; four pooled poorly-differentiated adenocarcinomas; from acute myelogenous leukemia; from chronic myelogenous leukemia; gall bladder; head_neck; heart; hepatocellular carcinoma, cell line; human retina; human skeletal muscle; hypemephroma; hypemephroma, cell line; hypothalamus, cell line; insulinoma; iris; islets of langerhans; kidney; kidney_tumor; large cell carcinoma; large cell carcinoma, undifferentiated; leiomyosarcoma; leiomyosarcoma cell line; lens; leukocyte; liver, lung; lung focal fibrosis; lung_normal; lung_tumor; lymph; marrow; metastatic chondrosarcoma; mixed (pool of 40 mas); mucoepidermoid carcinoma; muscle; muscle (skeletal); neuroblastoma cells; normal epithelium; normal head/neck tissue; optic nerve; osteosarcoma, cell line; ovary; ovary (pool of 3); pancreas; papillary serous ovarian metastasis; parathyroid; pheochromocytoma; pituitary; placenta; placenta_normal; pool; pooled brain, lung, testis; pooled colon, kidney, stomach; pooled lung and spleen; pooled pancreas and spleen; primary b-cells from tonsils (cell line); primary lung epithelial cells; primitive neuroectoderm; prostate; prostate_normal; prostate-tumor; purified pancreatic islet; renal cell adenocarcinoma; rpe and choroid; serous papillary tumor; skin; spleen; squamous cell carcinoma, poorly differentiated (4 pooled tumors, including primary and metastatic); stomach; subchondral bone; testis; testis_normal; thyroid; tonsil; two pooled squamous cell carcinomas; uterus; uterus_tumor; whole embryo.
Human Fragilis is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Human Fragilis 9-27/Leu13A sequence related to human Fragilis, i.e., human Fragilis 9-27 (also known as Leu13) is also identified. This sequence comprises a member of the Fragilis family of proteins.
Human Fragilis 9-27/Leu13 Nucleic Acid Sequence
Human Fragilis 9-27/Leu13 Amino Acid Sequence
Human Fragilis 9-27/Leu13 Genomic Location
Human Fragilis 9-27/Leu13 is located on chromosome 11.
Human Fragilis 9-27/Leu13 Diseases
Human Fragilis 9-27/Leu13 is expressed in a number of tissues, including: Stomach; adenocarcinoma; adenocarcinoma, cell line; adipose; adrenal cortico adenoma for cushing's syndrome; amnion normal; ascites; bone; bone marrow; brain; breast; cervical carcinoma cell line; chondrosarcoma; colon; colon ins; cord blood; ear; embryonal carcinoma; epid tumor; epitheloid carcinoma; eye; eye anterior segment; from acute myelogenous leukemia; from chronic myelogenous leukemia; germ cell; head neck; head normal; heart; human retina; hypothalamus, cell line; insulinoma; kidney; kidney tumor; large cell carcinoma; large cell carcinoma, undifferentiated; leiomios; leiomyosarcoma; leukocyte; liver; lung; lung normal; lymph; marrow; mixed (pool of 40 mas); mucoepidermoid carcinoma; muscle; muscle (skeletal); nervous tumor; normal head/neck tissue; nose; optic nerve; osteoarthritic cartilage; ovary; pancreas; papillary serous ovarian metastasis; parathyroid; pheochromocytoma; pituitary; placenta; placenta normal; pool; pooled; pooled brain, lung, testis; pooled pancreas and spleen; primary b-cells from tonsils (cell line); primitive neuroectoderm; prostate; prostate normal; purified pancreatic islet; retinal pigment epithelium sheets; rpe and choroid; skin; spleen; stomach; testis, cell line; testis normal; tonsil; trabecular meshwork; uterus; uterus tumor; whole embryo.
Human Fragilis 9-27/Leu13 is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 2Furthermore, we have identified several mouse sequences (namely, Fragilis 2, Fragilis 3, Fragilis 4, Fragilis 5 and Fragilis 6) which are related to Fragilis and comprise Fragilis family members.
Mouse Fragilis 2 Nucleic Acid Sequence
Mouse Fragilis 2 Amino Acid Sequence
Mouse Fragilis 2 Diseases
Mouse Fragilis 2 is expressed in a number of tissues, including: adult; branchial arches; colon; embryo; embryonal carcinoma; head; heart; hippocampus; liver; lymph; macrophage; mammary gland; mandible; muscle; pancreas; pool; pooled lung tumors; pooled mammary gland tumors; salivary gland; skin; small intestine; spleen; spontaneous tumor, metastatic to mammary. stem cell origin; stomach; subformical organ and postrema; t cell; testis; thymus; tumor, biopsy sample; tumor, gross tissue; tumor, metastatic to mammary; uterus; whole embryo.
Mouse Fragilis 2, as well as its human homologue, is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 3Mouse Fragilis 3 Nucleic Acid Sequence
Mouse Fragilis 3 Amino Acid Sequence
Mouse Fragilis 3 Genomic Location
Mouse Fragilis 3 is located on chromosome 16.
Mouse Fragilis 3 Diseases
Mouse Fragilis 3 is expressed in a number of tissues, including: adult; branchial round spermatids, pooled from multiple mice; testis.
Mouse Fragilis 3, as well as its human homologue, is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 4Mouse Fragilis 4 Nucleic Acid Sequence
Mouse Fragilis 4 Amino Acid Sequence
Mouse Fragilis 4 Diseases
Mouse Fragilis 4 is expressed in a number of tissues, including: bowel; cerebellum; colon; embryo; head; heart; kidney; liver; lung; lymph; macrophage; mammary gland; placenta; spleen; spontaneous tumor, metastatic to mammary. stem cell origin; stomach; t cell; thymus; tumor, biopsy sample; tumor, gross tissue; tumor, metastatic to mammary; vagina.
Mouse Fragilis 4, as well as its human homologue, is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 5Mouse Fragilis 5 Nucleic Acid Sequence
Mouse Fragilis 5 Amino Acid Sequence
Mouse Fragilis 5 Genomic Location
Mouse Fragilis 5 is located on chromosome 6.
Mouse Fragilis 5 Diseases
Mouse Fragilis 5 is expressed in a number of tissues, including: embryo; tumor, gross tissue.
Mouse Fragilis 5, as well as its human homologue, is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Mouse Fragilis 6Mouse Fragilis 6 Nucleic Acid Sequence
Mouse Fragilis 6 Amino Acid Sequence
Mouse Fragilis 6 Genomic Location
Mouse Fragilis 6 is located on chromosome 7.
Mouse Fragilis 6 Diseases
Mouse Fragilis 6 is expressed in a number of tissues, including: dbEST Library Tissue Type restricted to Spleen; cDNA sources: in vitro fertilized eggs; lymph; spinal cord; spleen.
Mouse Fragilis 6, as well as its human homologue, is therefore suitable for treatment or diagnosis of any disease of or associated with such tissues, in particular, cancer or tumours of such tissues.
Human Ifitm1, Ifitm2 Ifitm3 and ENSG142056Human Ifitm1, Ifitm2 Ifitm3
We disclose four homologues of Fragilis genes, located on Chromosome 11 (p15.5). This region is syntenic to the Fragilis family locus on mouse Chr 7 (
Three of these genes, Ifitm1 (9-27), Ifitm2 (1-8D) and Ifitm3 (1-8U), share 58-65% similarity to the fragilis gene cluster and are located within an 18 kb genomic stretch [AR Lewin, L E Reid, M McMahon, G R Stark, I M Kerr: Molecular analysis of a human interferon-inducible gene family. Eur J Biochem 1991, 199: 417-423].
They are responsive to type½ interferons and code for interferon induced transmembrane (Ifitm) proteins, involved in antiproliferative signalling and homotypic cell adhesion. See R L Friedman, S P Manley, M Meahon, I M Kerr, G R Stark: Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 1984, 38: 745-755; J M Kelly, C S Gilbert, G R Stark, I M Kerr: Differential regulation of interferon-induced mRNAs and c-myc mRNA by alpha- and gamma-interferons. Eur J Biochem 1985, 153:367-371; S S Evans, D B Lee, T Han, T B Tomasi, R L Evans: Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells. Blood 1990, 76 (12): 2583-2593; and S S Evans, R P Collea, J A Leasure, D B Lee: IFN-a induces homotypic adhesion and Leu-13 expression in human B lymphoid cells J Immunol 1993, 150: 736-747.
Interferon stimulable response elements (ISREs, GGAAAN(N)GAAAC) within the human Ifitm locus confer the responsiveness of the three human Ifitm genes to interferons (A R Lewin, L E Reid, M McMahon, G R Stark, I M Kerr: Molecular analysis of a human interferon-inducible gene family. Eur J Biochem 1991, 199: 417-423; L E Reid, A H Brasnett, C S Gilbert, A C G Porter, D R Gewert, G R Stark, I M Kerr: A single DNA response element can confer inducibility by both alpha- and gamma-interferons. Proc Natl Acad Sci USA 1989, 86: 840-844). Similar ISRE consensus sequences are also found within the Fragilis family cluster in the mouse, associated in particular with fragilis, fragilis 2 and fragilis5 (
Human ENSG142056
We further disclose the fourth gene, ENSG142056, a novel gene with two exons, is highly similar to mouse fragilis4 (83% DNA sequence similarity) and neighbours Ifitm2. The human Fragilis family homologues hence form a similar genomic cluster as the five Fragilis genes in the mouse.
Other Fragilis Homologues
We also identified two Fragilis family-like genes in cow (bovine 1-8U, bovine 9-27) and four genes in rat (P26376, JC1241, NP110460, AAD48010). While the rat genes have been annotated as putative interferon inducible, the two bovine genes that are similar to the human Ifitm genes, have been reported to respond to interferon signalling (D J Hayzer, E Brinson, M S Runge: A rat beta-interferon-induced mRNA: sequence characterization. Gene 1992, 117 (2): 227-228; 17. J K Pru, K J Austin, A L Haas, T R Hansen: Pregnancy and interferon-tau upregulate gene expression of members of the 1-8 family in the bovine uterus. Biol Reprod 2001, 65 (5): 1471-1480).
Functions of Human and StellaThe functions of human Stella and Fragilis are disclosed throughout this document. It is also expected that human Stella and Fragilis will broadly display the properties of their mouse counterparts. The functional homology of the human sequences disclosed here to their mouse Stella and Fragilis counterparts may be verified by a complementation test. An example of such a complementation test is set out in the following paragraphs.
Mouse mutants for Stella and Fragilis are created by gene knockout technology using ES cells, using standard techniques. Further details are provided in the Examples, particularly Examples 11 to 15. Human STELLA and FRAGILIS clones may be isolated by screening human genomic libraries, such as BAC libraries, or by nucleotide synthesis or PCR of a human cDNA library using the sequences disclosed here.
These BAC clones may then be introduced into the mouse genome by microinjection of the genomic clones into the oocyte or by transfection, lipofection or by using viral vectors, into ES cells. Transgenic mice carrying human homologues of STELLA and FRAGILIS are generated and crossed with mouse mutants for Stella or Fragilis. If the human homologue contains appropriate regulatory sequences and is therefore is expressed similarly to the mouse gene, it will overcome phenotypic deficiencies in mutant mice, if it is a true homologue. The wild type phenotype will be restored to the transgenic knockout mouse.
BAC clones may be modified to contain reporter genes such as GFP. When introduced into the mouse genome to generate transgenic mice, the reporter constructs can be used to verify whether the human homologue shows appropriate temporal and spatial patterns of expression. This expression analysis combined with the phenotypic rescue of mutants described above would verify if the human genes are the homologues of murine genes.
There are other ways in which murine and human genes can be tested for their effects on murine tumours. If the tumours are the result of gain of function, it would be possible to use RNAi or other antisense approaches to see if repression of modifies the phenotype. If tumours result from loss of function, transgenes (as BACs or under the control of appropriate tissue-specific regulatory sequences) can be introduced into cells or mice to check if they have an effect on tumours.
The procedure for BAC modifications and the generation of transgenic mice may employ the technique described in Developmental Biology, volume 236, 2001 by John, R M et al, or any other suitable technique.
Stella Diseases and Fragilis Associated DiseasesHuman Stella and Fragilis are expressed in a number of tissues and cells, and in particular in a number of tumour or cancerous cells and tissues, as described in further detail below. Accordingly, the Stella and/or Fragilis polypeptides, nucleic acids, antibodies, peptides, etc described here may be used in the diagnosis and/or treatment or prevention of any diseases associated with such tissues and cells, for example, tumours or cancers of the tissues and cells.
Examples of diseases associated with Stella and Fragilis are set out in the sections above relating to individual Stella and Fragilis sequences.
Particular further examples of such tumours and cancers include, but are not limited to, testis tumor, colon tumor, stomach, germ cell tumors, choriocarcinoma, lung, large cell carcinoma, uterus, and leiomyosarcoma. Alternatively or in addition, Stella and/or Fragilis associated diseases include any one or more of diseases of the following tissues, including: adenocarcinoma; adrenal cortico adenoma for cushing's syndrome; cervical carcinoma; Crohn's disease; embryonal carcinoma; endometrium adenocarcinoma; epitheloid carcinoma; poorly-differentiated adenocarcinomas; acute myelogenous leukemia; chronic myelogenous leukemia; hepatocellular carcinoma; hypernephroma; insulinoma; iris; kidney tumor, large cell carcinoma; large cell carcinoma, undifferentiated; leiomyosarcoma; lung focal fibrosis; metastatic chondrosarcoma; mucoepidermoid carcinoma; neuroblastoma; papillary serous ovarian metastasis; parathyroid; pheochrornocytoma; prostate tumor; serous papillary tumor; squamous cell carcinoma, preferably poorly differentiated.
Stella and Fragilis PolypeptidesIt will be understood that polypeptide sequences disclosed here are not limited to the particular sequences set forth in the sequence listing, or fragments thereof, or sequences obtained from Fragilis or Stella protein, but also include homologous sequences obtained from any source, for example related cellular homologues, homologues from other species and variants or derivatives thereof, provided that they have at least one of the biological activities of Stella or Fragilis (including related sequences such as Fragilis 2, 3, 4, 5 and 6), as the case may be.
This disclosure therefore encompasses variants, homologues or derivatives of the amino acid sequences set forth in this description, including in the sequence listings, as well as variants, homologues or derivatives of the amino acid sequences encoded by the nucleotide sequences disclosed here. In a preferred embodiment, the homologues and variants of Stella and Fragilis do not include the sequences disclosed in PCT/GB02/00215, in particular do not include the mouse Stella and Fragilis sequences set out in SEQ ID NO: 2 and 1 respectively. Furthermore, they preferably do not include any sequence portion of these genes previously disclosed without any indication of function, for example, AC006927.26.74831.117191 (Chromosome 12, p12.3). In particular, preferred embodiments do not comprise any EST (Expressed Sequence Tag) which may have been disclosed, for example, any one or more of the following ESTs: gi|953020|gb|AA300687.1|AA300687 EST13535 Testis tumor cDNA 5′ end; gi|8149136|gb|AW959452.1|AW959452 EST371522 MAGE resequences, MAGF cDNA (colon tumor); gi|10896747|gb|BF091037.1 |BF091037 MR3-SN0036-120900-007-h05 SN0036 cDNA (stomach normal); and gi|3367222|gb|AI066520.1|AI066520 ov17h01.x1 NCI_CGAP_GC3 cDNA clone IMAGE:1637617 3′ (pooled germ cell tumors)
In further preferred embodiments, the variants, homologues, etc of Stella and Fragilis do not include any one or more of the following ESTs: gi|2335869|gb|AA564230.1|AA564230 nk43h01.s1 NCI_CGAP_GC2 cDNA clone IMAGE:1016305 3′ (germ cell tumor); gi|3076239|gb|AA927342.1|AA927342 orn69e05.s1 NCI_CGAP_GC4 cDNA clone IMAGE:1552448 3′ (pooled germ cell tumors); gi|2959237|gb|AA864924.1|AA864924 oh44h08.s1 NCI_CGAP_GC4 cDNA clone IMAGE:1469535 3′ (pooled germ cell tumors); gi|9512276|gb|BE466414.1|BE466414 hz21d11.x1 NCI_CGAP_GC6 cDNA clone IMAGE:3208629 3′ (pooled germ cell tumors); gi|5837653|gb|AI990772.1|AI990772 ws23f02.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2498043 3′ (pooled germ cell tumors); gi|5741162|gb|AI948852.1|AI948852 wq37c09.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2473456 3′ (pooled germ cell tumors); gi|473-4510|gb|AI650531.1|AI650531 wa92b04.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2303599 3′ (pooled germ cell tumors); gi|5849349|gb|AW002433.1|AW002433 wu61h07.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2524573 3′ (pooled germ cell tumors); gi|5746921|gb|AI954611.1|AI954611 wq34b05.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2473137 3′ (pooled germ cell tumors); gi|4735956|gb|AI651977.1|AI651977 wb64h02.x1 NCI_CGAP_GC6 cDNA clone IMAGE:2310483 3′ (pooled germ cell tumors); gi|19763950|gb|BQ028671.1|BQ028671 UI-1-EE0-ayz-b-02-0-UI.sl; NCI_CGAP_P17 cDNA clone UI-1-EE0-ayz-b-02-0-UI 3′ (choriocarcinoma); gi|9890324|gb|BE619386.1|BE619386 601473204F1 NIH_MGC—68 cDNA clone IMAGE:3876145 5′ (lung, large cell carcinoma); gi|10344247|gb|BE888191.1|BE888191 601511709F1 NIH_MGC—71 cDNA clone IMAGE:3912866 5′ (uterus, leiomyosarcoma); and gi|1933621|gb|AA286758.1|AA286758 zs51a11.r1 NCI_CGAP_GCB1 cDNA clone IMAGE:700988 5′ (germinal center B cell).
HomologuesThe polypeptides disclosed include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof. Thus polypeptides also include those encoding homologues of Fragilis and/or Stella from other species including animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. More specifically, homologues include human homologues.
In the context of the present document, a homologous sequence or homologue is taken to include an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 30, preferably 50, 70, 90 or 100 amino acids with Fragilis or Stella, for example as shown in the sequence listing herein. In the context of this document, a homologous sequence is taken to include an amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level, preferably over at least 50 or 100, preferably 200, 300, 400 or 500 amino acids with the sequence of Fragilis or Stella, for example Fragilis (SEQ ID NO: 6) and Stella (SEQ ID NO: 4), or the Fragilis family member sequences Fragilis 9-27 (preferably comprising a sequence as shown in SEQ ID NO: 8), Fragilis 2 (preferably comprising a sequence as shown in SEQ ID NO: 10), Fragilis 3 (preferably comprising a sequence as shown in SEQ ID NO: 12), Fragilis 4 (preferably comprising a sequence as shown in SEQ ID NO: 14), Fragilis 5 (preferably comprising a sequence as shown in SEQ ID NO: 16) and Fragilis 6 (preferably comprising a sequence as shown in SEQ ID NO: 18).
Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present document it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.
However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package university of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
Variants and DerivativesThe terms “variant” or “derivative” in relation to the amino acid sequences as described here includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence. Preferably, the resultant amino acid sequence retains substantially the same activity as the unmodified sequence, preferably having at least the same activity as the Fragilis and/or Stella polypeptides shown in the sequence listings. Thus, the key feature of the sequences—namely that they are specific for PGCs and other pluripotent cells, such as ES or EG cells, and can serve as a marker for these cells in a cell population—is preferably retained.
Polypeptides having the amino acid sequence shown in the Examples, or fragments or homologues thereof may be modified for use in the methods and compositions described here. Typically, modifications are made that maintain the biological activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.
Natural variants of Fragilis and Stella are likely to comprise conservative amino acid substitutions. Conservative substitutions may be defined, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Polypeptides disclosed here and useful as markers also include fragments of the above mentioned full length polypeptides and variants thereof, including fragments of the sequences set out in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
Polypeptides also include fragments of the full length sequence of any of the Fragilis and/or Stella polypeptides. Preferably fragments comprise at least one epitope. Methods of identifying epitopes are well known in the art. Fragments will typically comprise at least 6 amino acids, more preferably at least 10, 20, 30, 50 or 100 amino acids.
Included are fragments comprising, preferably consisting of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150, or more residues from a Fragilis and/or Stella amino acid sequence.
Polypeptide fragments of the Stella/Fragilis proteins and allelic and species variants thereof may contain one or more (e.g. 5, 10, 15, or 20) substitutions, deletions or insertions, including conserved substitutions. Where substitutions, deletion and/or insertions occur, for example in different species, preferably less than 50%, 40% or 20% of the amino acid residues depicted in the sequence listings are altered.
Fragilis and Stella, and their fragments, homologues, variants and derivatives, may be made by recombinant means. However, they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. The proteins may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the function of the protein of interest sequence. Proteins may also be obtained by purification of cell extracts from animal cells.
The Fragilis and/or Stella polypeptides, variants, homologues, fragments and derivatives disclosed here may be in a substantially isolated form. It will be understood that such polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A Fragilis/Stella variant, homologue, fragment or derivative may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein.
The Fragilis/Stella polypeptides, variants, homologues, fragments and derivatives disclosed here may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide, etc to be detected. Suitable labels include radioisotopes, e.g. 125I, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides may be used in diagnostic procedures such as immunoassays to determine the amount of a polypeptide in a sample. Polypeptides or labelled polypeptides may also be used in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.
Fragilis/Stella polypeptides, variants, homologues, fragments and derivatives disclosed here, optionally labelled, my also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick. Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. Such polypeptides and kits may be used in methods of detection of antibodies to the polypeptides or their allelic or species variants by immunoassay.
Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.
The Fragilis/Stella polypeptides, variants, homologues, fragments and derivatives disclosed here may be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and homologues thereof in cell function, including their function in disease. For example, truncated or modified polypeptides may be introduced into a cell to disrupt the normal functions which occur in the cell. The polypeptides may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below). The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.
The use of appropriate host cells, such as insect cells or mammalian cells, is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products. Such cell culture systems in which the Fragilis/Stella polypeptides, variants, homologues, fragments and derivatives disclosed here are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides in the cell.
Fragilis/Stella Nucleic AcidsWe provide generally for a number of Fragilis and Stella nucleic acids, together with fragments, homologues, variants and derivatives thereof. These nucleic acid sequences preferably encode the polypeptide sequences disclosed here, and particularly in the sequence listings. Preferably, the polynucleotides comprise Stella and/or Fragilis nucleic acids, preferably selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 or fragments, homologues, variants and derivatives thereof.
In particular, we provide for nucleic acids which encode any of the Fragilis and/or Stella polypeptides disclosed here. Thus, the terms “GCR nucleic acid”, “Fragilis nucleic acid” and “Stella nucleic acid” should be construed accordingly. Preferably, however, such nucleic acids comprise any of the sequences set out as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 or a sequence encoding any of the corresponding polypeptides, and a fragment, homologue, variant or derivative of such a nucleic acid. The above terms therefore preferably should be taken to refer to these sequences.
In preferred embodiments, the Stella nucleic acid comprises a nucleic acid having a sequence set out in SEQ ID NO: 3 and the Fragilis nucleic acid comprises a nucleic acid having a sequence set out in SEQ ID NO: 5. However, also included are nucleic acids encoding any Fragilis family member, for example: Fragilis 9-27 (preferably a sequence as shown in SEQ ID NO: 7), Fragilis 2 (preferably a sequence as shown in SEQ ID NO: 9), Fragilis 3 (preferably a sequence as shown in SEQ ID NO: 11), Fragilis 4 (preferably a sequence as shown in SEQ ID NO: 13), Fragilis 5 (preferably a sequence as shown in SEQ ID NO: 15) and Fragilis 6 (preferably a sequence as shown in SEQ ID NO: 17).
As used here in this document, the terms “polynucleotide”, “nucleotide”, and nucleic acid are intended to be synonymous with each other. “Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.
It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
Variants, Derivatives and HomologuesThe polynucleotides described here may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present document, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides.
Where the polynucleotide is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the methods and compositions described here. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included.
The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleotides from or to the sequence providing the resultant nucleotide sequence is specific for pluripotent cells, preferably specific for PGCs, ES cells or EG cells. Most preferably, the resultant nucleotide sequence is specific for PGCs.
As indicated above, with respect to sequence identity, a “homologue” has preferably at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to the relevant sequence shown in the sequence listings.
More preferably there is at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity. Nucleotide homology comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and −9 for each mismatch. The default gap creation penalty is −50 and the default gap extension penalty is −3 for each nucleotide.
HybridisationWe further describe nucleotide sequences that are capable of hybridising selectively to any of the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
The term “hybridisation” as used herein shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction technologies.
Polynucleotides capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
The term “selectively hybridisable” means that the polynucleotide used as a probe is used under conditions where a target polynucleotide is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screened. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P.
Hybridisation conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.), and confer a defined “stringency” as explained below.
Maximum stringency typically occurs at about Tm-5° C. (5° C. below the Tm of the probe); high stringency at about 5° C. to 10° C. below Tm; intermediate stringency at about 10° C. to 20° C. below Tm; and low stringency at about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.
In a preferred aspect, we disclose nucleotide sequences that can hybridise to a Fragilis/Stella nucleic acid, or a fragment, homologue, variant or derivative thereof, under stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na3 Citrate pH 7.0}).
Where a polynucleotide is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present disclosure. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also disclosed and encompassed.
Polynucleotides which are not 100% homologous to the sequences disclosed here but fall within the disclosure can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells, including human cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of SEQ ID NOs: 1 or 3 under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of Fragilis and Stella.
The polynucleotides described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides as used herein. Preferred fragments are less than 500, 200, 100, 50 or 20 nucleotides in length.
Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
Nucleotide VectorsThe polynucleotides can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, we provide a method of making polynucleotides by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
Preferably, a polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term “operably linked” means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
Vectors may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
Control sequences operably linked to sequences encoding the protein include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term “promoter” is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
The promoter is typically selected from promoters which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of α-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the Rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
Expression of Stella and/or Fragilis Polypeptides
In order to express a biologically active Stella and/or Fragilis, the nucleotide sequences encoding Stella and/or Fragilis or homologues, variants, or derivatives thereof are inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
Methods which are well known to those skilled in the art are used to construct expression vectors containing sequences encoding Stella and/or Fragilis and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory Manual, ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.) and Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.).
A variety of expression vector/host systems may be utilized to contain and express sequences encoding Stella and/or Fragilis. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Any suitable host cell may be employed.
The “control elements” or “regulatory sequences” are those non-translated regions of the vector (i.e., enhancers, promoters, and 5′ and 3′ untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL), and the like, may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding Stella and/or Fragilis, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be selected depending upon the use intended for Stella and/or Fragilis. For example, when large quantities of Stella and/or Fragilis are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding Stella and/or Fragilis may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509), and the like. pOEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel (supra) and Grant et al. (1987; Methods Enzymol. 153:516-544).
In cases where plant expression vectors are used, the expression of sequences encoding Stella and/or Fragilis may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.).
An insect system may also be used to express Stella and/or Fragilis. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding Stella and/or Fragilis may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of Stella and/or Fragilis will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which Stella and/or Fragilis may be expressed. (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227.)
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding Stella and/or Fragilis may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing Stella and/or Fragilis in infected host cells. (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Thus, for example, the Stella and/or Fragilis proteins are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor expression, typically all 5′ and 3′ untranslated regions (UTRs) are removed from the receptor cDNA prior to insertion into a pCDN or pcDNA3 vector. The cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as negative controls. To isolate cell lines stably expressing the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resistant clones analyzed.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of about 6 kb 5 to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding Stella and/or Fragilis. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding Stella and/or Fragilis and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used, such as those described in the literature. (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding, and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
For long term, high yield production of recombinant proteins, stable expression is preferred. For example, cell lines capable of stably expressing Stella and/or Fragilis can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase genes (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in tk− or apr− cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt confers resistance to the aminoglycosides neomycin and G418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51.) Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding Stella and/or Fragilis is inserted within a marker gene sequence, transformed cells containing sequences encoding Stella and/or Fragilis can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding Stella and/or Fragilis under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells which contain the nucleic acid sequence encoding Stella and/or Fragilis and express Stella and/or Fragilis may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
The presence of polynucleotide sequences encoding Stella and/or Fragilis can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding Stella and/or Fragilis. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding Stella and/or Fragilis to detect transformants containing DNA or RNA encoding Stella and/or Fragilis.
A variety of protocols for detecting and measuring the expression of Stella and/or Fragilis, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on Stella and/or Fragilis is preferred, but a competitive binding assay may be employed. These and other assays are well described in the art, for example, in Hampton, R et al. (1990; Serological Methods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) and in Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding Stella and/or Fragilis include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding Stella and/or Fragilis, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding Stella and/or Fragilis may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be located in the cell membrane, secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode Stella and/or Fragilis may be designed to contain signal sequences which direct secretion of Stella and/or Fragilis through a prokaryotic or eukaryotic cell membrane. Other constructions may be used to join sequences encoding Stella and/or Fragilis to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences, such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.), between the purification domain and the Stella and/or Fragilis encoding sequence may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing Stella and/or Fragilis and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMIAC; described in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase cleavage site provides a means for purifying Stella and/or Fragilis from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).
Fragments of Stella and/or Fragilis may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Various fragments of Stella and/or Fragilis may be synthesized separately and then combined to produce the full length molecule.
Recombinant Stella and Fragilis ProteinsWe also provide for expression of Stella and Fragilis proteins, for example, recombinant proteins. These may be expressed by a number of methods as generally known in the art; the following provides an example of how this may be achieved.
Nucleotide sequences of Stella and Fragilis are cloned into a TRI-system vector (Qiagen). Stella sequence comprising the second codon onwards (i.e., an N terminal fragment of Stella without the first ATG codon) is cloned into a pQE vector using appropriate restriction enzyme sites, and according to the manufacturers instructions. QIAexpress pQE vectors enable high-level expression of 6×His-tagged proteins in E. coli.
A His tag is placed in the N terminal portion of the Stella gene. Recombinant protein is purified by affinity chromatography on a Ni-NTA column, according to manufacturer's instructions. The His tag is cleaved using a suitable protease.
Recombinantly expressed Stella and Fragilis protein are found to be biologically active.
Modulators, Agonists and AntagonistsThe methods and compositions described here rely, in some embodiments, on blocking, reducing, or increasing the activity of proteins such as Stella and Fragilis, for example, in methods of treating or preventing a disease such as cancer. In general, the methods employ modulators of Stella and/or Fragilis activity or expression.
Agents which are capable of increasing the activity of a the Stella and/or Fragilis protein are referred to as agonists of that activity. Similarly, antagonists reduce the activity of the relevant protein.
The term “antagonist”, as used in the art, is generally taken to refer to a compound which binds to an enzyme and inhibits the activity of the enzyme. The term as used here, however, is intended to refer broadly to any agent which inhibits the activity of a molecule, not necessarily by binding to it. Accordingly, it includes agents which affect the expression of a protein such as Stella or Fragilis, or the biosynthesis of a regulatory molecule, or the expression of modulators of the activity of the Stella or Fragilis. The specific activity which is inhibited may be any activity which is exhibited by, or characteristic of, the enzyme or molecule, for example, any activity of Stella and/Fragilis as the case may be, for example, a signal transduction activity or PGC specification activity.
The antagonist may bind to and compete for one or more sites on the relevant molecule preferably, the catalytic site of the enzyme. Preferably, such binding blocks the interaction between the molecule and another entity (for example, the interaction between a enzyme and its substrate). However, the antagonist need not necessarily bind directly to a catalytic site, and may bind for example to an adjacent site, another protein (for example, a protein which is complexed with the enzyme) or other entity on or in the cell, so long as its binding reduces the activity of the enzyme or molecule.
Where antagonists of a enzyme such as a enzyme are concerned, an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme. In addition, whole or fragments of a substrate generated natively or by peptide synthesis may be used to compete with the substrate for binding sites on the enzyme. Alternatively, or in addition, an immunoglobulin (for example, a monoclonal or polyclonal antibody) capable of binding to the enzyme may be used. The antagonist may also include a peptide or other small molecule which is capable of interfering with the binding interaction. Other examples of antagonists are set forth in greater detail below, and will also be apparent to the skilled person.
Blocking the activity of an inhibitor of the relevant Stella and/or Fragilis protein may also be achieved by reducing the level of expression of the protein or an inhibitor in the cell. For example, the cell may be treated with antisense compounds, for example oligonucleotides having sequences specific to the Stella and/or Fragilis mRNA. The level of expression of pathogenic forms of adhesion proteins may also be regulated this way.
In general, agonists, antagonists and modulators comprise agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate. An agent may be in solution or in suspension (e.g., in crystalline, colloidal or other particulate form). The agent may be in the form of a monomer, dimer, oligomer, etc, or otherwise in a complex.
The terms “modulator”, “antagonist” and “agent” are also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signalling molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g. a yeast artificial chromosome) or a part thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. Small molecules, including inorganic and organic chemicals, which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented, are also included. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
In a particular embodiment, the technique of RNA interference (RNAi) may be used to abolish or knock out or reduce gene activity, for example, Stella and/or Fragilis activity. The overall strategy is to prepare double stranded RNA (dsRNA) specific to each gene of interest and to transfect this into a cell of interest to inhibit the expression of the particular gene.
The following protocol may be used: a sample of PCR product is analysed by horizontal gel electrophoresis and the DNA purified using a Qiagen QiaQuick PCR purification kit. 1 μg of DNA is used as the template in the preparation of gene specific single stranded RNA using the Ambion T7 Megascript kit. Single stranded RNA is produced from both strands of the template and is purified and immediately annealed by heating to 90 degrees C. for 15 mins followed by gradual cooling to room temperature overnight. A sample of the dsRNA is analysed by horizontal gel electrophoresis, and introduced into the relevant cell by conventional means.
Identifying Modulators, Agonists and AntagonistsModulators, agonists and antagonists of Stella and/or Fragilis activity or expression may be identified by any means known in the art. Putative such molecules may be identified by their binding to Stella and/or Fragilis, in an assay which detects binding between Stella (or Fragilis as the case may be) and the putative molecule.
Assays to detect modulators, agonists or antagonists typically involve detecting modulation of any activity of Stella and/or Fragilis in the presence, optionally together with detection of modulation of activity in the absence, of a candidate molecule. The assays involve contacting a candidate molecule with Stella or Fragilis, whether in the form of a polypeptide, a nucleic acid encoding the polypeptide, or a cell, organelle, extract, or other material comprising such, with a candidate modulator. The relevant activity of Stella or Fragilis (as described below) may be detected, to establish whether the presence of the candidate modulator has any effect. Promoter binding assays to detect candidate modulators which bind to and/or affect the transcription or expression of Stella and/or Fragilis may also be used. Candidate modulators may then be chosen for further study, or isolated for use. Details of such screening procedures are well known in the art, and are for example described in, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9).
The screening methods described here preferably employ in vivo assays, although they may be configured for in vitro use. In vivo assays generally involve exposing a cell comprising Stella and/or Fragilis to the candidate molecule. In in vitro assays, Stella and/or Fragilis is exposed to the candidate molecule, optionally in the presence of other components, such as crude or semi-purified cell extract, or purified proteins. Where in vitro assays are conducted, these preferably employ arrays of candidate molecules (for example, an arrayed library). In vivo assays are preferred. Preferably, therefore, the Stella and/or Fragilis is comprised in a cell, preferably heterologously. Such a cell is preferably a transgenic cell, which has been engineered to express Stella and/or Fragilis.
It will be appreciated that any component of a cell comprising Stella and/or Fragilis may be employed, such as an organelle. A preferred embodiment utilises a nuclear preparation, e.g., comprising a cell nucleus which comprises Stella and/or Fragilis as described. The nuclear preparation may comprise one or more nuclei, which may be permeabilised or semi-permeabilised, by detergent treatment, for example.
Thus, in a specific embodiment, an assay format may include the following: a multiwell microtitre plate is set up to include one or more cells expressing Stella and/or Fragilis in each well; individual candidate molecules, or pools of candidate molecules, may be added to individual wells and modulation of Stella and/or Fragilis activity measured. Where pools are used, these may be subdivided in to further pools and tested in the same manner.
Alternatively or in addition to the assay methods described above, “subtractive” procedures may also be used to identify modulators, agonists or antagonists of Stella and/or Fragilis. Under such “subtractive” procedures, a plurality of molecules is provided, which comprises one or more candidate molecules capable of functioning as a modulator (e.g., cell extract, nuclear extract, library of molecules, etc), and one or more components is removed, depleted or subtracted from the plurality of molecules. The “subtracted” extract, etc, is then assayed for activity, by exposure to a cell comprising Stella and/or Fragilis (or a component thereof) as described.
Thus, for example, an ‘immunodepletion’ assay may be conducted to identify such modulators as follows. A nuclear extract may be prepared from a pluripotent cell, for example, a pluripotent EG/ES cell. The extract may be depleted or fractionated to remove putative modulators, such as by use of immunodepletion with appropriate antibodies. If the extract is depleted of a modulator, it will lose the ability to affect Stella and/or Fragilis function or activity or expression. A series of subtractions and/or depletions may be required to identify the modulators, agonists or antagonists.
It will also be appreciated that the above “depletion” or “subtraction” assay may be used as a preliminary step to identify putative modulatory factors for further screening. Furthermore, or alternatively, the “depletion” or “subtraction” assay may be used to confirm the modulatory activity of a molecule identified by other means (for example, a “positive” screen as described elsewhere in this document) as a putative modulator.
Candidate molecules subjected to the assay and which are found to be of interest may be isolated and further studied. Methods of isolation of molecules of interest will depend on the type of molecule employed, whether it is in the form of a library, how many candidate molecules are being tested at any one time, whether a batch procedure is being followed, etc.
The candidate molecules may be provided in the form of a library. In a preferred embodiment of the invention, more than one candidate molecule is screened simultaneously. A library of candidate molecules may be generated, for example, a polypeptide library, a nucleic acid library, a library of compounds (such as a combinatorial library), a library of antisense molecules such as antisense DNA or antisense RNA, an antibody library etc, by means known in the art. Such libraries are suitable for high-throughput screening. Different cells comprising Stella and/or Fragilis may be exposed to individual members of the library, and expression of the reporter or reporters detected. Array technology may be employed for this purpose. The cells may be spatially separated, for example, in wells of a microtitre plate.
In a preferred embodiment, a small molecule library is employed. By this term, we refer to a library of molecules, whose molecular weights are individually less than about 50 kDa. In particular embodiments, small molecule libraries comprise molecules having molecular weights preferably less than about 30 kDa, more preferably less than about 15 kDa, most preferably less than 10 kDa or so. Such “small molecule” libraries may contain polypeptides, small peptides, for example, peptides of 20 amino acids or fewer, for example, 15, 10 or 5 amino acids, simple compounds, etc.
Alternatively or in addition, a combinatorial library, as described in further detail below, may be screened for modulators, antagonists or agonists of Stella and/or Fragilis.
Any of the activities of Stella and/or Fragilis may be used as the basis of the assay. In particular, cellular activities mediated by Stella and/or Fragilis may be assayed to identify antagonists. For example, Fragilis family members are responsible for homotypic adhesion between cells, and effects of the putative antagonist or agonist on adhesion activity mediated by Fragilis family members may be assayed using for example cell adhesion assays as known in the art. Furthermore, we show that Fragilis family members are capable of lengthening cell cycle times; accordingly, the cell cycle period may be assayed in the presence and absence of a candidate molecule to identify antagonists or agonists of Fragilis activity.
LibrariesLibraries of candidate molecules, such as libraries of polypeptides or nucleic acids, may be employed in the methods and compositions described here. Such libraries are exposed to the nucleic acid encoding the reporter (or cell comprising such a nucleic acid), for the detection of expression of the reporter.
Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith (1990 supra), have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen. The nucleotide sequences encoding the VH and VL regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E. coli and as a result the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pIII or pVIII). Alternatively, antibody fragments are displayed externally on lambda phage capsids (phagebodies). An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al. (1990) supra; Kang et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Natl. Acad Sci U.S.A., 88:10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al. (1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins and Winter (1992) J. Immunol., 22: 867; Marlcs et al., 1992, J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313, incorporated herein by reference). Such techniques may be modified if necessary for the expression generally of polypeptide libraries.
One particularly advantageous approach has been the use of scFv phage-libraries (Bird, R. E., et al. (1988) Science 242: 423-6, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991) supra; Marks et al. (1991) supra; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et al. (1992) supra). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Refinements of phage display approaches are also known, for example as described in WO96/06213 and WO92/01047 (Medical Research Council et al.) and WO97/08320 (Morphosys, supra), which are incorporated herein by reference.
Alternative library selection technologies include bacteriophage lambda expression systems, which may be screened directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et al. (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and are of use in the invention. These expression systems may be used to screen a large number of different members of a library, in the order of about 106 or even more. Other screening systems rely, for example, on direct chemical synthesis of library members. One early method involves the synthesis of peptides on a set of pins or rods, such as described in WO84/03564. A similar method involving peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, is described in U.S. Pat. No. 4,631,211 and a related method is described in WO92/00091. A significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide, so as to facilitate identification of the amino acid sequence of each library member. These improved bead-based methods are described in WO93/06121.
Another chemical synthesis method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface in a manner that places each distinct library member (e.g., unique peptide sequence) at a discrete, predefined location in the array. The identity of each library member is determined by its spatial location in the array. The locations in the array where binding interactions between a predetermined molecule (e.g., a receptor) and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location. These methods are described in U.S. Pat. No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991) Science, 251: 767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26: 271.
Other systems for generating libraries of polypeptides or nucleotides involve the use of cell-free enzymatic machinery for the in vitro synthesis of the library members. In one method, RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar technique may be used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843). In a similar way, in vitro translation can be used to synthesise polypeptides as a method for generating large libraries. These methods which generally comprise stabilised polysome complexes, are described further in WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536. Alternative display systems which are not phage-based, such as those disclosed in WO95/22625 and WO95/11922 (Affymax) use the polysomes to display polypeptides for selection. These and all the foregoing documents also are incorporated herein by reference.
The library may in particular comprise a library of zinc fingers; zinc fingers are known in the art and act as transcription factors. Suitable zinc finger libraries are disclosed in, for example, WO 96/06166 and WO 98/53057. Construction of zinc finger libraries may utilise rules for determining interaction with specific DNA sequences, as disclosed in for example WO 98/53058 and WO 98/53060. Zinc fingers capable of interacting specifically with methylated DNA are disclosed in WO 99/47656. The above zinc finger libraries may be immobilised in the form of an array, for example as disclosed in WO 01/25417. Accordingly, preferred molecules capable of altering the potency of a cell include zinc fingers.
Combinatorial LibrariesLibraries, in particular, libraries of candidate molecules, may suitably be in the form of combinatorial libraries (also known as combinatorial chemical libraries).
A “combinatorial library”, as the term is used in this document, is a collection of multiple species of chemical compounds that consist of randomly selected subunits. Combinatorial libraries may be screened for molecules which are capable of causing expression of the reporter, and optionally of altering the potency of the cell.
Various combinatorial libraries of chemical compounds are currently available, including libraries active against proteolytic and non-proteolytic enzymes, libraries of agonists and antagonists of G-protein coupled receptors (GPCRs), libraries active against non-GPCR targets (e.g., integrins, ion channels, domain interactions, nuclear receptors, and transcription factors) and libraries of whole-cell oncology and anti-infective targets, among others. A comprehensive review of combinatorial libraries, in particular their construction and uses is provided in Dolle and Nelson (1999), Journal of Combinatorial Chemistry, Vol 1 No 4, 235-282. Reference is also made to Combinatorial peptide library protocols (edited by Shmuel Cabilly, Totowa, N.J.: Humana Press, c 1998 Methods in Molecular Biology; v. 87). Specific combinatorial libraries and methods for their construction are disclosed in U.S. Pat. No. 6,168,914 (Campbell™, et al), as well as in Baldwin et al. (1995), “Synthesis of a Small Molecule Library Encoded with Molecular Tags,” J. Am. Chem. Soc. 117:5588-5589, and in the references mentioned in those documents.
In a preferred embodiment, the combinatorial library which is screened is one which is designed to potentially include molecules which interact with a component of the cell to influence gene expression. For example, combinatorial libraries against chromatin structural proteins may be screened. Other libraries which are useful for this embodiment include combinatorial libraries against histone modification enzymes (e.g., histone acetylation or histone methylation enzymes), or DNA modification, for example, DNA methylation or demethylation.
Further references describing chemical combinatorial libraries, their production and use include those available from the URL http://www.netsci.org/Science/Combichem/, including The Chemical Generation of Molecular Diversity. Michael R. Pavia, Sphinx Pharmaceuticals, A Division of Eli Lilly (Published July, 1995); Combinatorial Chemistry: A Strategy for the Future—MDL Information Systems discusses the role its Project Library plays in managing diversity libraries (Published July, 1995); Solid Support Combinatorial Chemistry in Lead Discovery and SAR Optimization, Adnan M. M. Mjalli and Barry E. Toyonaga, Ontogen Corporation (Published July, 1995); Non-Peptidic Bradykinin Receptor Antagonists From a Structurally Directed Non-Peptide Library. Sarvajit Chakravarty, Babu J. Mavunkel, Robin Andy, Donald J. Kyle*, Scios Nova Inc. (Published July, 1995); Combinatorial Chemistry Library Design using Pharmacophore Diversity Keith Davies and Clive Briant, Chemical Design Ltd. (Published July, 1995); A Database System for Combinatorial Synthesis Experiments—Craig James and David Weininger, Daylight Chemical Information Systems, Inc. (Published July, 1995); An Information Management Architecture for Combinatorial Chemistry, Keith Davies and Catherine White, Chemical Design Ltd. (Published July, 1995); Novel Software Tools for Addressing Chemical Diversity, R. S. Pearlman, Laboratory for Molecular Graphics and Theoretical Modeling, College of Pharmacy, University of Texas (Published June/July, 1996); Opportunities for Computational Chemists Afforded by the New Strategies in Drug Discovery: An Opinion, Yvonne Connolly Martin, Computer Assisted Molecular Design Project, Abbott Laboratories (Published June/July, 1996); Combinatorial Chemistry and Molecular Diversity Course at the University of Louisville: A Description, Arno F. Spatola, Department of Chemistry, University of Louisville (Published June/July, 1996); Chemically Generated Screening Libraries: Present and Future. Michael R. Pavia, Sphinx Pharmaceuticals, A Division of Eli Lilly (Published June/July, 1996); Chemical Strategies For Introducing Carbohydrate Molecular Diversity Into The Drug Discovery Process. Michael J. Sofia, Transcell Technologies Inc. (Published June/July, 1996); Data Management for Combinatorial Chemistry. Maryjo Zaborowski, Chiron Corporation and Sheila H. DeWitt, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company (Published November, 1995); and The Impact of High Throughput Organic Synthesis on R&D in Bio-Based Industries, John P. Devlin (Published March, 1996).
Techniques in combinatorial chemistry are gaining wide acceptance among modern methods for the generation of new pharmaceutical leads (Gallop, M. A. et al., 1994, J. Med. Chem. 37:1233-1251; Gordon, E. M. et al., 1994, J. Med. Chem. 37:1385-1401.). One combinatorial approach in use is based on a strategy involving the synthesis of libraries containing a different structure on each particle of the solid phase support, interaction of the library with a soluble receptor, identification of the ‘bead’ which interacts with the macromolecular target, and determination of the structure carried by the identified ‘bead’ (Lam, K. S. et al., 1991, Nature 354:82-84). An alternative to this approach is the sequential release of defined aliquots of the compounds from the solid support, with subsequent determination of activity in solution, identification of the particle from which the active compound was released, and elucidation of its structure by direct sequencing (Salmon, S. E. et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712), or by reading its code (Kerr, J. M. et al., 1993, J. Am. Chem. Soc. 115:2529-2531; Nikolaiev, V. et al., 1993, Pept. Res. 6:161-170; Ohlmeyer, M. H. J. et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926).
Soluble random combinatorial libraries may be synthesized using a simple principle for the generation of equimolar mixtures of peptides which was first described by Furka (Furka, A. et al., 1988, Xth International Symposium on Medicinal Chemistry, Budapest 1988; Furka, A. et al., 1988, 14th International Congress of Biochemistry, Prague 1988; Furka, A. et al., 1991, Int. J. Peptide Protein Res. 37:487-493). The construction of soluble libraries for iterative screening has also been described (Houghten, R. A. et al. 1991, Nature 354:84-86). K. S. Lam disclosed the novel and unexpectedly powerful technique of using insoluble random combinatorial libraries. Lam synthesized random combinatorial libraries on solid phase supports, so that each support had a test compound of uniform molecular structure, and screened the libraries without prior removal of the test compounds from the support by solid phase binding protocols (Lam, K. S. et al., 1991, Nature 354:82-84).
Thus, a library of candidate molecules may be a synthetic combinatorial library (e.g., a combinatorial chemical library), a cellular extract, a bodily fluid (e.g., urine, blood, tears, sweat, or saliva), or other mixture of synthetic or natural products (e.g., a library of small molecules or a fermentation mixture).
A library of molecules may include, for example, amino acids, oligopeptides, polypeptides, proteins, or fragments of peptides or proteins; nucleic acids (e.g., antisense; DNA; RNA; or peptide nucleic acids, PNA); aptamers; or carbohydrates or polysaccharides. Each member of the library can be singular or can be a part of a mixture (e.g., a compressed library). The library may contain purified compounds or can be “dirty” (i.e., containing a significant quantity of impurities).
Commercially available libraries (e.g., from Affymetrix, ArQule, Neose Technologies, Sarco, Ciddco, Oxford Asymmetry, Maybridge, Aldrich, Panlabs, Pharmacopoeia, Sigma, or Tripose) may also be used with the methods described here.
In addition to libraries as described above, special libraries called diversity files can be used to assess the specificity, reliability, or reproducibility of the new methods. Diversity files contain a large number of compounds (e.g., 1000 or more small molecules) representative of many classes of compounds that could potentially result in nonspecific detection in an assay. Diversity files are commercially available or can also be assembled from individual compounds commercially available from the vendors listed above.
AntibodiesSpecific antagonists of Stella and/or Fragilis, which may be used to regulate the activity of these proteins (for example, for methods of treating or preventing diseases such as cancer) may include antibodies against the protein(s).
Antibodies, as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab′ and F(ab′)2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small-fragments, such as Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
The antibodies according described here are especially indicated for the detection of PGCs and other pluripotent cells, such as ES or EG cells. Accordingly, they may be altered antibodies comprising an effector protein such as a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo or in vitro. Such labels may be radioactive labels or radiopaque labels, such as metal particles, which are readily visualisable within an embryo or a cell mass. Moreover, they may be fluorescent labels or other labels which are visualisable on tissue samples.
Recombinant DNA technology may be used to improve the antibodies as described here. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [EP 0 239 400].
Antibodies may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.
Therefore, we disclose a process for the production of an antibody comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said antibody protein, and isolating said protein.
Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incorporated herein by reference.
The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of PGCs or other pluripotent cells, such as ES or EG cells, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with Fragilis or Stella, or fragments thereof, or with Protein-A.
Hybridoma cells secreting the monoclonal antibodies are also provided. Preferred hybridoma cells are genetically stable, secrete monoclonal antibodies of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.
Also included is a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to Fragilis and/or Stella, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with a one or more Fragilis or Stella polypeptides, or antigenic fragments thereof, antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example spleen cells of Balb/c mice immunised with Fragilis and/or Stella are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 10 and 107 and 108 cells expressing Fragilis and/or Stella and a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30% to about 50% polyethylene glycol of a molecular weight around 4000. After the fusion the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
Recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to Fragilis and/or Stella as described hereinbefore are also disclosed. By definition such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to Fragilis and/or Stella can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
Also disclosed are recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed to Fragilis and/or Stella fused to a human constant domain g, for example γ1, γ2, γ3 or γ4, preferably γ1 or γ4. Likewise recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to Fragilis and/or Stella fused to a human constant domain κ or λ, preferably κ are also disclosed.
In another embodiment, we disclose recombinant DNAs coding for a recombinant-polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule.
The DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.
Anti-Peptide Stella and Fragilis AntibodiesAnti-peptide antibodies may be produced against Stella and Fragilis peptide sequences. The sequences chosen may be based on the mouse sequences as follow:
Corresponding sequences from human Stella and Fragilis may be chosen for use in eliciting anti-peptide antibodies from immunised animals. Antibodies may be produced by injection into rabbits, and other conventional means, as described in for example, Harlow and Lane (supra).
Antibodies are checked by Elisa assay and by Western blotting, and used for immunostaining as described in the Examples.
Therapeutic PeptidesThe polypeptides disclosed here, for example, Stella and Fragilis polypeptides, may be used therapeutically for treatment of various diseases, including cancer, in the form of peptides comprising any portion of their sequence.
Where such Stella and/or Fragilis peptides are used therapeutically, it is preferred to use peptides that do not consist solely of naturally-occurring amino acids but which have been modified, for example to reduce immunogenicity, to increase circulatory half-life in the body of the patient, to enhance bio-availability and/or to enhance efficacy and/or specificity.
A number of approaches have been used to modify peptides for therapeutic application. One approach is to link the peptides or proteins to a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG)—see for example U.S. Pat. Nos. 5,091,176, 5,214,131 and U.S. Pat. No. 5,264,209.
Replacement of naturally-occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify the Stella and/or Fragilis peptides.
Another approach is to use bi-functional crosslinkers, such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-2 pyridyldithio) propionamido]hexanoate, and sulfosuccinimidyl 6-[3-(2 pyridyldithio) propionamido]hexanoate (see U.S. Pat. No. 5,580,853).
It may be desirable to use derivatives of the peptides disclosed here which are conformationally constrained. Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide. Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.
The active conformation of the peptide may be stabilised by a covalent modification, such as cyclization or by incorporation of γ-lactam or other types of bridges. For example, side chains can be cyclized to the backbone so as create a L-γ-lactam moiety on each side of the interaction site. See, generally, Hruby et al., “Applications of Synthetic Peptides,” in Synthetic Peptides: A User's Guide: 259-345 (W. H. Freeman & Co. 1992). Cyclization also can be achieved, for example, by formation of cystine bridges, coupling of amino and carboxy terminal groups of respective terminal amino acids, or coupling of the amino group of a Lys residue or a related homologue with a carboxy group of Asp, Glu or a related homologue. Coupling of the alpha-amino group of a polypeptide with the epsilon-amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken. See Wood and Wetzel, 1992, Int'l J. Peptide Protein Res. 39, 533-39.
Another approach described in U.S. Pat. No. 5,891,418 is to include a metal-ion complexing backbone in the peptide structure. Typically, the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the co-ordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a co-ordination number of four to six. The nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulfur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities. In addition, the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino. The peptide construct can be either linear or cyclic, however a linear construct is typically preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly which has four nitrogen atoms (an N4 complexation system) in the back bone that can complex to a metal ion with a co-ordination number of four.
A further technique for improving the properties of therapeutic peptides is to use non-peptide peptidomimetics. A wide variety of useful techniques may be used to elucidating the precise structure of a peptide. These techniques include amino acid sequencing, x-ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, computer-assisted molecular modelling, peptide mapping, and combinations thereof. Structural analysis of a peptide generally provides a large body of data which comprise the amino acid sequence of the peptide as well as the three-dimensional positioning of its atomic components. From this information, non-peptide peptidomimetics may be designed that have the required chemical functionalities for therapeutic activity but are more stable, for example less susceptible to biological degradation. An example of this approach is provided in U.S. Pat. No. 5,811,512.
Techniques for chemically synthesising therapeutic peptides are described in the above references and also reviewed by Borgia and Fields, 2000, TibTech 18, 243-251 and described in detail in the references contained therein.
Transgenic AnimalsWe further describe transgenic animals capable of expressing natural or recombinant Stella and/or Fragilis, or a homologue, variant or derivative, at elevated or reduced levels compared to the normal expression level. Included are transgenic animals (“Stella knockout”s or “Fragilis knockout”s) which do not express functional Stella and/or Fragilis, as the case may be. The Stella and Fragilis knockouts may arise as a result of functional disruption of the Stella and/or Fragilis gene or any portion of that gene, including one or more loss of function mutations, including a deletion or replacement, of the Stella and/or Fragilis gene. The mutations include single point mutations, and may target coding or non-coding regions of Stella and/or Fragilis.
Preferably, such a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent. Most preferably the transgenic animal is a mouse or a rat. Such transgenic animals may be used in screening procedures to identify agonists and/or antagonists of Stella and/or Fragilis, as well as to test for their efficacy as treatments for diseases in vivo.
Mice which are null for Stella and/or Fragilis may be used for various purposes. For example, transgenic animals that have been engineered to be deficient in the production of Stella and/or Fragilis may be used in assays to identify agonists and/or antagonists of Stella and/or Fragilis. One assay is designed to evaluate a potential drug (aa candidate ligand or compound) to determine if it produces a physiological response in the absence Stella and/or Fragilis. This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response.
Tissues derived from the Stella and/or Fragilis knockout animals may be used in binding assays to determine whether the potential drug (a candidate ligand or compound) binds to Stella or Fragilis, as the case may be. Such assays can be conducted by obtaining a first Stella and/or Fragilis preparation from the transgenic animal engineered to be deficient in Stella and/or Fragilis production and a second Stella and/or Fragilis preparation from a source known to bind any identified ligands or compounds. In general, the first and second preparations will be similar in all respects except for the source from which they are obtained. For example, if brain tissue from a transgenic animal (such as described above and below) is used in an assay, comparable brain tissue from a normal (wild type) animal is used as the source of the second preparation. Each of the preparations is incubated with a ligand known to bind to Stella and/or Fragilis, both alone and in the presence of the candidate ligand or compound. Preferably, the candidate ligand or compound will be examined at several different concentrations.
The extent to which binding by the known ligand is displaced by the test compound is determined for both the first and second preparations. Tissues derived from transgenic animals may be used in assays directly or the tissues may be processed to isolate Stella and/or Fragilis proteins, which are themselves used in the assays. A preferred transgenic animal is the mouse. The ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described in further detail above).
Furthermore, antagonists of Stella and/or Fragilis may be identified by administering candidate compounds, etc, to wild type animals expressing functional Stella and/or Fragilis, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of Stella and/or Fragilis function.
Methods for generating non-human transgenic animal are known in the art, and are described in further detail in the Examples below. Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
In an exemplary embodiment, the transgenic non-human animals described here are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used to produce transgenic animals are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor.
Introduction of the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. For example, the Stella or Fragilis transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s). Following introduction of the transgene construct into the fertilized egg, the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is also included. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
The progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.
The litters of transgenically altered mammals can be assayed after birth for the incorporation of the construct into the genome of the offspring. Preferably, this assay is accomplished by hybridizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny. Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity.
For the purposes of this document, a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism. Generally, the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes. Thus, the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism. Generally, a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
In addition to similar biological considerations, physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters. The physical effects of addition must not be so great as to physically destroy the viability of the zygote. The biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
The number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. There will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
The transgenic animals produced in accordance the methods described here will include exogenous genetic material. As set out above, the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a Stella and/or Fragilis protein. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
It will be appreciated that it is possible to manipulate the control elements (promoters or enhancers) to regulate the spatial or temporal expression, or both, of Stella or Fragilis (as the case may be). For example, specific control elements may be deleted from the endogenous Stella and/or Fragilis locus so that expression is restricted to only certain tissues. Alternatively, it is possible to prepare transgenes which only contain one, some, or more, of the control elements. Transgenic animals made this way for Stella and/or Fragilis and having properties of ectopic expression, temporally or spatially, or both, will be useful for investigation of Stella and/or Fragilis gene function.
Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, K (1988) Science 240:1468-1474.
We also provide non-human transgenic animals, where the transgenic animal is characterized by having an altered Stella and/or Fragilis gene, preferably as described above, as models for Stella or Fragilis function, as the case may be. Alterations to the gene include deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof. The transgenic animals may be either homozygous or heterozygous for the alteration. The animals and cells derived therefrom are useful for screening biologically active agents that may modulate Stella and/or Fragilis function. The screening methods are of particular use for determining the specificity and action of potential therapies for Stella and/or Fragilis associated diseases, as described above. The animals are useful as a model to investigate the role of Stella and/or Fragilis proteins in the body.
Another aspect pertains to a transgenic animal having a functionally disrupted endogenous Stella or Fragilis gene, or both, but which also carries in its genome, and expresses, a transgene encoding a heterologous Stella and/or Fragilis protein (i.e., a Stella and/or Fragilis gene from another species). Preferably, the animal is a mouse and the heterologous Stella or Fragilis is a human Stella or Fragilis. An animal, or cell lines derived from such an animal, which has been reconstituted with human Stella and/or Fragilis, can be used to identify agents that inhibit human Stella and/or Fragilis in vivo and in vitro. For example, a stimulus that induces signalling through human Stella and/or Fragilis can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured. An agent that inhibits human Stella and/or Fragilis in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.
We also provide for a Stella and/or Fragilis deficient transgenic non-human animal (a “Stella/Fragilis knock-out” or a “Stella/Fragilis null”). Such an animal is one which expresses lowered or no Stella/Fragilis activity, preferably as a result of an endogenous Stella or Fragilis (as the case may be) genomic sequence being disrupted or deleted. The endogenous Stella or Fragilis genomic sequence may be replaced by a null allele, which may comprise non-functional portions of the wild-type Stella/Fragilis sequence. For example, the endogenous Stella/Fragilis genomic sequence may be replaced by an allele of Stella/Fragilis comprising a disrupting sequence which may comprise heterologous sequences, for example, reporter sequences and/or selectable markers. Preferably, the endogenous Stella/Fragilis genomic sequence in a Stella/Fragilis knock-out mouse is replaced by an allele of Stella or Fragilis in which one or more, preferably all, of the coding sequences is replaced by such a disrupting sequence, preferably a lacZ sequence and a neomycin resistance sequence. Preferably, the genomic Stella/Fragilis sequence which is functionally disrupted comprises a mouse Stella/Fragilis genomic sequence.
Preferably, such an animal expresses no Stella or Fragilis activity, or both. More preferably, the animal expresses no activity of the Stella or Fragilis proteins shown in the sequence listings. Stella/Fragilis knock-outs may be generated by various means known in the art, as described in further detail below. A specific description of the construction of a Stella knock-out mouse is disclosed in Example 20 et seq below.
We further disclose a nucleic acid construct for functionally disrupting a Stella/Fragilis gene in a host cell. The nucleic acid construct comprises: a) a non-homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first Stella/Fragilis gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second Stella/Fragilis gene sequence, the second Stella/Fragilis gene sequence having a location downstream of the first Stella/Fragilis gene sequence in a naturally occurring endogenous Stella/Fragilis gene. Additionally, the first and second homology regions are of sufficient length for homologous recombination between the nucleic acid construct and an endogenous Stella/Fragilis gene in a host cell when the nucleic acid molecule is introduced into the host cell. In a preferred embodiment, the non-homologous replacement portion comprises an expression reporter, preferably including lacZ and a positive selection expression cassette, preferably including a neomycin phosphotransferase gene operatively linked to a regulatory element(s).
Another aspect pertains to recombinant vectors into which the nucleic acid construct described above has been incorporated. Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous Stella/Fragilis gene of the host cell, resulting in functional disruption of the endogenous Stella/Fragilis gene. The host cell can be a mammalian cell that normally expresses Stella/Fragilis from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell. Further development of an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous Stella/Fragilis gene produces a transgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the Stella/Fragilis gene disruption in their genome. Animals that carry the Stella/Fragilis gene disruption in their germline can then be selected and bred to produce animals having the Stella/Fragilis gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the Stella/Fragilis gene disruption.
Detection of Pluripotent Cells in Cell PopulationsPolynucleotide probes or antibodies as described here may be used for the detection of pluripotent cells such as primordial germ cells (PGCs), stem cells such as embryonic stem (ES) and embryonic germ (EG) cells in cell populations. As used herein, a “cell population” is any collection of cells which may contain one or more PGCs, ES or EG cells. Preferably, the collection of cells does not consist solely of PGCs, but comprises at least one other cell type.
Cell populations comprise embryos and embryo tissue, but also adult tissues and tissues grown in culture and cell preparations derived from any of the foregoing.
Polynucleotides as described here may be used for detection of Fragilis and Stella transcripts in PGCs or other pluripotent cells, such as ES or EG cells, by nucleic acid hybridisation techniques. Such techniques include PCR, in which primers are hybridised to Fragilis and/or Stella transcripts and used to amplify the transcripts, to provide a detectable signal; and hybridisation of labelled probes, in which probes specific for an unique sequence in the Fragilis and/or Stella transcript are used to detect the transcript in the target cells.
As noted hereinbefore, probes may be labelled with radioactive, radiopaque, fluorescent or other labels, as is known in the art.
The antibodies may also be used to detect Fragilis and/or Stella. Fragilis, in particular, possesses an extracellular domain which may be targeted by an anti-Fragilis antibody and detected at the cell surface. Alternatively, intracellular scFv may be used to detect Fragilis and/or Stella within the cell.
Particularly indicated are immunostaining and FACS techniques. Suitable fluorophores are known in the art, and include chemical fluorophores and fluorescent polypeptides, such as GFP and mutants thereof (see WO 97/28261). Chemical fluorophores may be attached to immunoglobulin molecules by incorporating binding sites therefor into the immunoglobulin molecule during the synthesis thereof.
Preferably, the fluorophore is a fluorescent protein, which is advantageously GFP or a mutant thereof. GFP and its mutants may be synthesised together with the immunoglobulin or target molecule by expression therewith as a fusion polypeptide, according to methods well known in the art. For example, a transcription unit may be constructed as an in-frame fusion of the desired GFP and the immunoglobulin or target, and inserted into a vector as described above, using conventional PCR cloning and Ligation techniques.
Antibodies may be labelled with any label capable of generating a signal. The signal may be any detectable signal, such as the induction of the expression of a detectable gene product Examples of detectable gene products include bioluminescent polypeptides, such as luciferase and GFP, polypeptides detectable by specific assays, such as β-galactosidase and CAT, and polypeptides which modulate the growth characteristics of the host cell, such as enzymes required for metabolism such as HIS3, or antibiotic resistance genes such as G418. In a preferred aspect, the signal is detectable at the cell surface. For example, the signal may be a luminescent or fluorescent signal, which is detectable from outside the cell and allows cell sorting by FACS or other optical sorting techniques.
Preferred is the use of optical immunosensor technology, based on optical detection of fluorescently-labelled antibodies. Immunosensors are biochemical detectors comprising an antigen or antibody species coupled to a signal transducer which detects the binding of the complementary species (Rabbany et al., 1994 Crit Rev Biomed Eng 22:307-346; Morgan et al., 1996 Clin Chem 42:193-209). Examples of such complementary species include the antigen Zif 268 and the anti-Zif 268 antibody. Immunosensors produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood (Robinson 1991 Biosens Dioelectron 6:183-191). The sensitivity of immunosensors makes them ideal for situations requiring speed and accuracy (Rabbany et al., 1994 Crit Rev Biomed Eng 22:307-346).
Detection techniques employed by immunosensors include electrochemical, piezoelectric or optical detection of the immunointeraction (Ghindilis et al., 1998 Biosens Bioelectron 1:113-131). An indirect immunosensor uses a separate labelled species that is detected after binding by, for example, fluorescence or luminescence (Morgan et al., 1996 Clin Chem 42:193-209). Direct immunosensors detect the binding by a change in potential difference, current, resistance, mass, heat or optical properties (Morgan et al., 1996 Clin Chem 42:193-209). Indirect immunosensors may encounter fewer problems due to non-specific binding (Attridge et al., 1991 Biosens Bioelecton 6:201-214; Morgan et al., 1996 Clin Chem 42:193-209).
Prophylactic and Therapeutic MethodsWe provide methods of treating an abnormal conditions related to both an excess of and insufficient amounts of Stella and/or Fragilis activity. Examples of these include the Stella associated diseases and Fragilis associated diseases disclosed above.
If the activity of Stella and/or Fragilis is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of a relevant molecule to the Stella and/or Fragilis, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
In another approach, where Stella and/or Fragilis act by binding a ligand soluble forms of Stella and/or Fragilis polypeptides still capable of binding the ligand in competition with endogenous Stella and/or Fragilis may be administered. Typical embodiments of such competitors comprise fragments of the Stella and/or Fragilis polypeptide.
In still another approach, expression of the gene encoding endogenous Stella and/or Fragilis can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Alternatively, oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
For treating abnormal conditions related to an under-expression of Stella and/or Fragilis and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates Stella and/or Fragilis, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of Stella and/or Fragilis by the relevant cells in the subject.
For example, a polynucleotide as described in this document may be engineered for expression in a replication defective retroviral vector. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a Stella and/or Fragilis polypeptide such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
Formulation and AdministrationPeptides and polypeptides, such as the Stella and/or Fragilis peptides and polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art. We further describe pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions.
Polypeptides and other compounds may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/or localize, in the form of salves, pastes, gels and the like.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as “gene therapy” as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
Pharmaceutical CompositionsWe also provide a pharmaceutical composition comprising administering a therapeutically effective amount of the polypeptide, polynucleotide, peptide, vector or antibody (such as a Stella and/or Fragilis polypeptide, etc) and optionally a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition as described here may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
VaccinesAnother embodiment relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the Stella and/or Fragilis polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from Stella and/or Fragilis associated disease.
Yet another embodiment relates to a method of inducing immunological response in a mammal which comprises delivering a Stella and/or Fragilis polypeptide via a vector directing expression of a Stella and/or Fragilis polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
A further embodiment relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a Stella and/or Fragilis polypeptide wherein the composition comprises a Stella and/or Fragilis polypeptide or Stella and/or Fragilis gene. The vaccine formulation may further comprise a suitable carrier.
Since the Stella and/or Fragilis polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Vaccines may be prepared from one or more polypeptides or peptides as described here.
The preparation of vaccines which contain an immunogenic polypeptide(s) or peptide(s) as active ingredient(s), is known to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
Further examples of adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).
Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al2O3 basis). Conveniently, the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.
After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4° C., or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension Reconstitution is preferably effected in buffer
Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
The polypeptides described here may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
AdministrationTypically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The pharmaceutical and vaccine compositions as disclosed here may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Typically, each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
The term “administered” includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term “administered” includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
The term “co-administered” means that the site and time of administration of each of for example, the polypeptide and an additional entity such as adjuvant are such that the necessary modulation of the immune system is achieved. Thus, whilst the polypeptide and the adjuvant may be administered at the same moment in time and at the same site, there may be advantages in administering the polypeptide at a different time and to a different site from the adjuvant. The polypeptide and adjuvant may even be delivered in the same delivery vehicle—and the polypeptide and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled.
The Stella and/or Fragilis polypeptide, polynucleotide, peptide, nucleotide, antibody etc and optionally an adjuvant may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
The vaccine composition and pharmaceutical compositions described here may be administered by a number of different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra-vaginal, urethral or ocular administration.
The vaccines and pharmaceutical compositions described here may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, may be 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
Diagnostic AssaysWe describe the use of Stella and/or Fragilis polynucleotides and polypeptides (as well as homologues, variants and derivatives thereof) for use in diagnosis as diagnostic reagents or in genetic analysis. Nucleic acids complementary to or capable of hybridising to Stella and/or Fragilis nucleic acids (including homologues, variants and derivatives), as well as antibodies against Stella and/or Fragilis polypeptides are also useful in such assays.
We provide for a natural variant of Stella and/or Fragilis polypeptide or nucleic acid, and the use of such a natural variant in diagnosis of Stella and/or Fragilis associated disease. Stella and/or Fragilis polymorphisms may include differences at the nucleic acid level, which may or may not reflect differences in the amino acid level. Preferably, such Stella and/or Fragilis variants or mutants are such that they include changes in the amino acid level. However, we also disclose Stella and/or Fragilis polymorphisms which occur in non-coding regions, for example, expression control regions such as promoters and enhancers.
Polymorphisms in Stella and/or Fragilis include deletions of one or more nucleic acids, insertions of one or more nucleic acids, inversions, etc. Preferably, Stella and/or Fragilis polymorphisms comprise single nucleotide polymorphisms.
Polymorphisms in Stella and/or Fragilis may be identified by comparing sequences at the appropriate level (whether nucleic acid or protein) between individuals in a population. Differences in sequences may be reflected in different physical properties, and techniques for detecting these may rely on detection of changes in physical properties. For example, single nucleotide polymorphisms may be detected as restriction fragment length polymorphisms (i.e., difference in susceptibility to digestion by a restriction enzyme). Furthermore, SNPS may affect the migration or mobility of a nucleic acid fragment or protein fragment in a gel.
Non-coding polymorphisms in Stella and/or Fragilis may be identified by sequencing non-coding regions of Stella and/or Fragilis. For example, control regions of the Stella and/or Fragilis gene, such as enhancers and promoters may be sequenced to identify polymorphisms. The effect of such non-coding polymorphisms on the expression level of Stella and/or Fragilis may be determined by constructing transgenic mice (as described below) comprising the mutant Stella and/or Fragilis sequences, or by generating expression constructs and transfection into cell lines. In each case, the expression level of Stella and/or Fragilis is detected, by RT-PCR or antibody Western staining, to determine the effect of the mutation in the control of expression of Stella and/or Fragilis. Useful Stella and/or Fragilis polymorphisms are those which modulate the level of expression, whether by up-regulation or down-regulation of Stella and/or Fragilis levels.
Accordingly, we provide for a variant or mutant or polymorphism in a non-coding region of Stella and/or Fragilis, preferably in a control region of Stella and/or Fragilis, preferably in a promoter and/or enhancer of Stella and/or Fragilis, which is capable of modulating the level of expression of Stella and/or Fragilis in an organism. We also provide for a set of two or more of such mutants or variants or polymorphisms, preferably non-coding polymorphisms. We also provide for the use of such variants or polymorphisms or sets of variants to identify nucleic acid and/or amino acid positions, in which changes to such positions affect the level of expression of Stella and/or Fragilis. We also provide for a transgenic animal comprising a variant or mutant or polymorphism of Stella and/or Fragilis, preferably, a non-coding polymorphism.
Detection of a mutated form of the Stella and/or Fragilis gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of Stella and/or Fragilis. Individuals carrying mutations in the Stella and/or Fragilis gene (including control sequences) may be detected at the DNA level by a variety of techniques.
For example, DNA may be isolated from a patient and the DNA polymorphism pattern of Stella and/or Fragilis determined. The identified pattern is compared to controls of patients known to be suffering from a disease associated with over-, under- or abnormal expression of Stella and/or Fragilis. Patients expressing a genetic polymorphism pattern associated with Stella and/or Fragilis associated disease may then be identified. Genetic analysis of the Stella and/or Fragilis gene may be conducted by any technique known in the art. For example, individuals may be screened by determining DNA sequence of a Stella and/or Fragilis allele, by RFLP or SNP analysis, etc. Patients may be identified as having a genetic predisposition for a disease associated with the over-, under-, or abnormal expression of Stella and/or Fragilis by detecting the presence of a DNA polymorphism in the gene sequence for Stella and/or Fragilis or any sequence controlling its expression.
Patients so identified can then be treated to prevent the occurrence of Stella and/or Fragilis associated disease, or more aggressively in the early stages of Stella and/or Fragilis associated disease to prevent the further occurrence or development of the disease. Stella and/or Fragilis associated diseases include any cancer, for example, as described above.
We further disclose a kit for the identification of a patient's genetic polymorphism pattern associated with Stella and/or Fragilis associated disease. The kit includes DNA sample collecting means and means for determining a genetic polymorphism pattern, which is then compared to control samples to determine a patient's susceptibility to Stella and/or Fragilis associated disease. Kits for diagnosis of a Stella and/or Fragilis associated disease comprising Stella and/or Fragilis polypeptide and/or an antibody against such a polypeptide (or fragment of it) are also provided.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. In a preferred embodiment, the DNA is obtained from blood cells obtained from a finger prick of the patient with the blood collected on absorbent paper. In a further preferred embodiment, the blood is collected on an AmpliCard™ (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF).
The DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis Oligonucleotide DNA primers that target the specific polymorphic DNA region within the genes of interest may be prepared so that in the PCR reaction amplification of the target sequences is achieved. RNA or cDNA may also be used as templates in similar fashion. The amplified DNA sequences from the template DNA may then be analyzed using restriction enzymes to determine the genetic polymorphisms present in the amplified sequences and thereby provide a genetic polymorphism profile of the patient. Restriction fragments lengths may be identified by gel analysis. Alternatively, or in conjunction, techniques such as SNP (single nucleotide polymorphisms) analysis may be employed.
Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled Stella and/or Fragilis nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, eg., Myers et al, Science (1985)230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method. See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotides probes comprising the Stella and/or Fragilis nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M. Chee et al., Science, Vol 274, pp 610-613 (1996)).
Single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci. USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control Stella and/or Fragilis nucleic acids may be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
The diagnostic assays offer a process for diagnosing or determining a susceptibility to Stella and/or Fragilis associated diseases, for example, as described above.
The presence of Stella and/or Fragilis polypeptides and nucleic acids may be detected in a sample. Thus, infections and diseases as listed above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the Stella and/or Fragilis polypeptide or Stella and/or Fragilis mRNA. The sample may comprise a cell or tissue sample from an organism suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal Stella and/or Fragilis expression, including spatial or temporal changes in level or pattern of expression. The level or pattern of expression of Stella and/or Fragilis in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression in a normal organism as a means of diagnosis of disease.
In general therefore, we describe a method of detecting the presence of a nucleic acid comprising a Stella and/or Fragilis nucleic acid in a sample, by contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid. For example, the nucleic acid probe may specifically bind to the Stella and/or Fragilis nucleic acid, or a portion of it, and binding between the two detected; the presence of the complex itself may also be detected. Furthermore, we disclose a method of detecting the presence of a Stella and/or Fragilis polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide. This may conveniently be achieved by monitoring the presence of a complex formed between the antibody and the polypeptide, or monitoring the binding between the polypeptide and the antibody. Methods of detecting binding between two entities are known in the art, and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.
Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a Stella and/or Fragilis, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
We also describe a diagnostic kit for a disease or susceptibility to a Stella and/or Fragilis associated disease (including an infection). The diagnostic kit comprises a Stella and/or Fragilis polynucleotide or a fragment thereof; a complementary nucleotide sequence; a Stella and/or Fragilis polypeptide or a fragment thereof, or an antibody to a Stella and/or Fragilis polypeptide.
Further Aspects of the InventionWe provide a nucleic acid molecule which is at least 90% homologous to SEQ ID NO: 3 and a nucleic acid molecule which is at least 75% homologous to SEQ ID NO: No. 5.
We disclose polynucleotides which comprise a contiguous stretch of nucleotides from SEQ ID NO: 3 or SEQ ID NO: 5, or any of SEQ ID NOs: 7, 9, 11, 13, 15 and 17, or of a sequence at least 90% homologous thereto. Advantageously, this stretch of contiguous nucleotides is 50 nucleotides in length, preferably 40, 35, 30, 25, 20, 15 or 10 nucleotides in length.
The human genes Fragilis and Stella encode novel polypeptides, the sequences of which are set forth in SEQ ID NO: 4 and SEQ ID NO: 6. Other Fragilis genes are set out in SEQ ID NOs: 8, 10, 12, 14, 16 and 18. We therefore disclose polypeptides encoded by the nucleic acids described here, as well as nucleic acids encoding any of the polypeptides disclosed here. Preferably, the polypeptides have the sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 6.
EXAMPLESThe following Examples 1 to 9 relate to cloning and characterisation of rodent Fragilis and Stella genes, and are included here for reference.
Example 1 Identification of Murine Genes Specific to the Earliest Population of Primordial Germ Cells (PGCs) by Single Cell cDNA Differential ScreeningA method for single cell analysis is developed to identify genes that are involved in the specification of the germ cell lineage, which results in the establishment of a founder population of Primordial Germ Cells (PGCs). It is determined that the lineage specification of PGCs accompanies the expression of a unique set of genes, which are not expressed in somatic cells.
The method for the identification of the genes is mainly based on the differential screening of the libraries made from single cells from day 7.25 mouse embryonic fragments that contain PGCs. The single cell cDNA differential screen was originally described by Brady and Iscove (1993), and subsequently modified by Cathaline Dulac and Richard Axel which resulted in the successful identification of the pheromone receptor genes from rat (Dulac, C. and Axel, 1995). The method of Axel's group is employed, with slight modifications as described.
Construction of Single Cell cDNAs from Embryonic Fragment Bearing the Earliest Population of PGCs
In the mouse, the earliest population of the PGCs is reported to consist of alkaline phosphatase positive cluster of some 40 cells, at the base of the emerging allantois at day 7.25 of gestation (Ginsburg, M., Snow, M. H. L., and McLaren, A. (1990)). The precise location of the PGC cluster in the inbred 129Sv and C57BL/6 strain is determined by microscopy using both whole-mount alkaline phosphatase staining and semi-thin sections stained by methylene blue. The earliest stage at which a cluster of PGcs can be detected is at the Late Streak stage (Downs, K. M., and Davies, T. (1993)), when a distinctively stained population of cells is found just beneath an epithelial lining from which the allantoic bud appears. This region is at the border between the extraembryonic and embryonic tissues just posterior to and above the most proximal part of the primitive streak. The cluster persists at this position at least until Early/Mid Bud stage. In the inbred 129Sv strain, the PGC cluster is found to contain a slightly larger number of the cells, which are more tightly packaged than in the C57BL/6 strain. The 129Sv strain is used for subsequent experiments, as a better recovery of the earliest PGCs is obtained.
129Sv embryos are isolated at E7.5 in DMEM plus 10% FCS buffered with 25 mM HEPES at room temperature and the developmental stage of each embryo is determined under a dissection microscope. The precise developmental stage can differ substantially even amongst embryos within the same litter. Embryos that are at the no bud or early bud (allantoic) stage are chosen for further dissection, which in part is dictated by the ease of identification of the region containing PGCs as seen under the dissection microscope. The fragment that is expected to contain the PGC cluster is cut out very precisely by means of solid glass needles. This region is dissociated it into single cells using 0.25% trypsin-1 mM EGTAJPBS treatment at 37° C. for 10 min, followed by gentle pipetting with a mouth pipette. The dissected fragment usually contained between 250-300 cells. The procedure for cell dispersal with this gentle procedure left the visceral endoderm layer remained as an intact cellular sheet.
We picked single cells randomly from the cell suspension by a mouth pipette and put individual single cells (but avoiding generating air bubbles), into a thin-walled PCR tube containing 4 μl of ice-cold cell lysis buffer (50 mM Tris-HCl pH8.3, 75 mM KCl, 3 mM MgCl2, 0.5% NP40, containing 80 ng/ml pd(T)24, 5 μg/ml prime RNase inhibitor, 324 U/ml RNA guard, and 10 mM each of dATP, dCTP, dGTP, and dTTP). The volume of medium carried with the single cell is less than 0.5 μl. The tube is briefly centrifuged to ensure that the cell is indeed in the lysis buffer. During each separate experiment, we picked a total of 19 single cells, and left one tube without a cell, to serve as a negative control for the PCR amplification procedure. All the cells that are collected in tubes are kept on ice before starting the subsequent procedure.
The cells are lysed by incubating the tubes at 65° C. for 1 min, and then kept at room temperature for 1-2 min to allow the oligo dT to anneal the to RNA. First-strand cDNA synthesis is initiated by adding 50 U of Moloney murine leukaemia virus (MMLV) and 0.5 U of avian myeloblastosis virus (AMV) reverse transcriptase followed by incubation for 15 min at 37° C. The reverse transcriptases are inactivated for 10 min at 65° C. This reverse transcription reaction is restricted to 15 min, which allows the synthesis of relatively uniform size cDNAs of between 500 base-1000 bases in length from the C termini. This enables the subsequent PCR amplification to be fairly representative.
Next, in order to add the poly A tail to the 5 prime end of the synthesised first-strand cDNA, 4.5 μl of 2× tailing buffer (200 mM potassium cacodylate pH7.2, 4 mM CoCl2, 0.4 mM DTT, 200 mM dATP containing 10 U of terminal transferase) is added to the reaction followed by incubation for 15 min at 37° C. The samples are heat inactivated for 10 min at 65° C. The reaction now contained synthesised cDNAs bearing poly T tail at their C termini and poly A stretch at their N termini, ready for the amplification by the PCR using the specific primer.
The contents of each tube is brought to 100 μl with a solution made of 10 mM Tris-HCl pH8.3, 50 mM KCl, 2.5 mM MgCl2, 100 μg/ml bovine serum albumin, 0-05% Triton-X 100, 1 mM of dATP, dCTP, dGTP, dTTP, 10 U of Taq polymerase, and 5 μg of the AL1 primer. The AL1 sequence is ATT GGA TCC AGG CCG CTC TGG ACA AAA TAT GAA TCC (T)24. The PCR amplification is performed according to the following schedule: 94° C. for 1 min, 42° C. for 2 min, and 72° C. for 6 min with 10 s extension per cycle for 25 cycles. Five additional units of Taq polymerase are added before performing 25 more cycles with the same programme but without the extension time. Each tube at this point contains amplified cDNA products derived from a single cell. The protein contents of the solution are extracted by phenol/chloroform treatment, and the amplified cDNAs are precipitated by ethanol and eventually suspended in 100 μl of TE pH8.0. 5 μl of the cDNA solution is run on a 1.5% agarose gel to check the success of the amplification. Most of the samples show a very intense ‘smeared’ band ranging mainly between 500 bp to 1200 bp, indicating the efficient amplification of the single cell cDNA. Only the successfully amplified samples are used for the subsequent ‘cell typing’ analysis.
Example 2 Identification of PGCs by Examination of the Expression of Marker GenesThe embryonic fragment which is excised theoretically contains three major components: the allantoic mesoderm, PGCs, and extraembryonic mesoderm surrounding PGCs. In order to identify the single cell cDNA of PGC origin amongst these samples, positive and negative selection of the constructed cDNAs is performed, by examining the expression of four marker genes (BMP4, TNAP, Hoxb1, and Oct4), which are known to be either expressed or repressed in various cell types in this region.
At the No/Early Bud stage, BMP4 is reported to be expressed in the emerging allantois and mesodermal components of the developing amnion, chorion, and visceral yolk sac (Lawson, K. A., Dunn, N. R., Roelen, B. A. J., Zeinstra, L. M., Davis, A. M., Wright, C. V. E., Korving, J. P. W. F. M., and Hogan, B. L. M. (1999)). The boundary of BMP4 expression is very sharp, and the expression is completely excluded in the mesodermal region beneath the epithelial lining continuous from the amnionic mesoderm where the putative PGCs are determined. Therefore, BMP4 is used as a negative marker for the selection. Primer pairs are designed for amplifying the C terminal portion of BMP4 (5′: GCC ATA CCT TGA CCC GCA GAA G, 3′: AAA TGG CAC TCA GTT CAG TGG G). The PCR amplification is performed using 0.5 μl of the cDNA solution as a template according to the following schedule: 95° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min for 20 cycles. Among 83 samples tested, 57 samples show the expected size of bands, indicating expression of BMP4 these single cells. These samples are considered to be of allantoic mesodermal origin, and therefore excluded from amongst the candidates representing cells of PGC origin.
The expression of tissue non-specific alkaline phosphatase (TNAP), which has long been used as an early marker for PGCs (Ginsburg, M., Snow, M. H. L., and McLaren, A. (1990)), is then examined. Primer pairs are designed (5′: CCC AAA GCA CCT TAT TTT TCT ACC, 3′: TTG GCG AGT CTC TGC AAT TGG) and the same PCR reaction as above is performed. Amongst the 26 samples, 22 samples are judged to be positive for TNAP. From the alkaline phosphatase staining of the sectioned embryos, it is known that the somatic cells surrounding PGCs also express some amount of TNAP, although the level of expression is slightly lower than that in PGCs. Therefore, amongst these 22 positive samples there should be still be cells destined to become somatic cells as well as PGCs.
One of the genes known to be expressed in the totipotent PGCs but not in somatic cells is Oct4 (Yoem, Y. II., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., and Scholer, H. R. (1996)). To examine the possibility that Oct4 can be used as a marker to distinguish PGCs from somatic cells at this stage, Oct4 expression is checked in the 22 samples by PCR (5′: CAC TCT ACT CAG TCC CTT TTC, 3′: TGT GTC CCA GTC TTT ATT TAA G). All the 22 samples express Oct4 at comparable levels, indicating that the somatic cells at this stage are still actively transcribing Oct4 RNA.
The amount of expression of TNAP is quantitated in 22 samples by Southern blot analysis (reverse northern blot analysis). Given the fairly representative amplification of the single cell method, confirmed by amplifying single ES cell cDNA, Southern blot analysis allows semi-quantitative measurement of the amount of the genes expressed in the original single cells, although it does not serve as a perfect indicator of cell identity. However, as a result of this TNAP analysis, 10 samples out of 22 show relatively stronger bands at an equivalent level, while the remaining 12 samples exhibit weaker signals. These results indicate that these 22 samples can be divided at least into two groups, one with stronger TNAP expression (therefore from putative PGCs) and the other with weaker TNAP.
The possibility that somatic cells surrounding PGCs start to express Hoxb1, while PGCs do not (personal communication from Dr. Kirstie Lawson) is also examined. Primer pairs are designed (5′: AAC TCA TCA GAG GTC GAA GGA, 3′: CGG TGC TAT TOT AAG GTC TGC) and the same PCR reaction as above is performed. Among the 22 samples tested, 12 are positive, and more importantly, these 12 samples perfectly match the ones which show weaker TNAP signals, by Southern blot analysis.
Taking all these results into consideration, it is concluded that 10 samples out of 83, which are Oct4 (+), TNAP (++), BMP4 (−), and Hoxb1 (−), are of PGC origin. This ratio (10/83) is reasonable, considering the number of the founding population of PGCs as 40 and the number of cells in the fragment as 250-300.
Example 3 Differential Screening of Single Cell cDNA LibrariesAs the efficiency of the amplification of cDNA differs in each tube, it is very important to select the samples with the most efficiently amplified cDNA for the construction of libraries. The amplification of six different genes (ribosomal protein S112, intermediate filament protein vimentin, β tubulin-5, α actin, Oct4, E-cadherin) is examined in the 10 PGC candidate samples, by Southern blot analysis. Judging from the overall profile of the amplification of all these six genes, three cDNA preparations are selected for the construction of libraries.
To obtain the maximum amount of double strand cDNA, an extension step is performed with 5 μl of cell cDNA in 100 μl of the PCR buffer described as above (including 1 μl of AmpliTaq) according to the following schedule: 94° C. for 5 min, 42° C. for 5 min, 72° C. for 30 min. The solution is extracted by phenol/chloroform treatment, and the amplified cDNAs are precipitated by ethanol, suspended in TE, and completely digested with EcoRI. The PCR primer and excess amount of dNTPs are removed by QIAGEN PCR Purification Kit, and all the purified cDNAs are run on a 2% low melting agarose gel. cDNAs above 500 bp are cut and purified by QIAGEN Gel Purification Kit. The purified eDNAs are precipitated by ethanol and suspended in TE and ligated into λ ZAP II vector arms. The ligated vector is packaged, titered and the ratio of the successfully ligated clones is monitored by amplifying the inserts with T3 and T7 primers from 20 plaques. More than 95% of the phage are found to contain inserts.
The representation of the three genes, ribosomal protein S12, β tubulin-5, Oct4, is quantitated by screening 5000 plaques, and the library of the best quality among the three (S12 0.62%, β tubulin 0.4%, Oct4 0.5%) is used for the differential screening. As a comparison partner with the PGC probe, one of the most efficiently amplified surrounding somatic cell cDNA (Oct4 (+), TNAP(+/−), BMP(−), and Hoxb1(+)) is selected by the similar Southern blot analysis.
The library is plated at a density of 1000 plaques per 15 cm dish to obtain large plaques (2 mm diameter) and two duplicate lifts are taken using Hybond N+ filters from Amersham. The filters are prehybridized at 65° C. in 0.5M sodium phosphate buffer (pH7.3) containing 1% bovine serum albumin and 4% SDS. We prepared the cell cDNA probes by reamplifying for 10 cycles 111 of the original cell cDNA into 501 of total reaction with the AL1 primer, in the absence of cold dCTP and with 100 μCi of newly received 32PdCTP, followed by the purification using Amersham Nick™ Spin Column. The filters are hybridised for at least 16 hrs with 1.0×107 cpm/ml (The first filter is hybridised with somatic cell probe and the second filter is hybridised with the PGC probe). After the hybridisation, the filters are washed three times at 65° C. in 0.5×SSC, 0.5% SDS and exposed to X ray films until the appropriate signal is obtained (usually one to two days).
The positive plaques in the two duplicate filters are compared very carefully. Among 5000 plaques screened, 280 are picked as candidates representing the differentially expressed genes. The inserts of all the 280 plaques are amplified with T3 and T7 primers, run on 1.5% gels, and double sandwich Southern blotted. Each membrane is hybridised with the PGC and somatic cell probe, respectively, using the same conditions as the screening. 38 clones amongst the 280 are selected as differentially expressed genes. These clones are next hybridised with the second PGC and somatic cell cDNA probes, which resulted in 20 clones out of 38 to be common in both PGC cDNAs but they are either not included or less abundant in both somatic cell cDNAs. The sequences of all the 20 clones are determined.
Genes Highly Specific to the Earliest Population of PGCs
The 20 clones represent 11 different genes (two clones appear two times, one clone appears three times, and one clone appears 6 times). To further stringently check the specificity of expression, primer pairs are designed for these 11 clones and their expression checked in 10 different single PGC-candidate cDNAs and 10 different single somatic cell cDNAs by PCR. Two of them show highly specific expression to PGC cDNAs.
The first gene, Fragilis (Germ cell restricted-1, Fragilis), encodes a 137 amino acid protein with a predicted molecular weight of 15.0 kD. The nucleic acid sequence of mouse Fragilis is set out in SEQ ID NO: 2.
Fragilis, comprises two transmembrane domains, the amino and carboxy terminus ends being located outside the cell. Fragilis is a newly discovered member of the interferon (IFN)-inducible transmembrane protein family (Deblandre, G. A. et al. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 270, 23860-23866 (1995); Friedman, R. L., Manly, S. P., McMahon, M., Kerr, I. M. & Stark, G. R. Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 38, 745-755 (1984) and is detected in expressed sequence tags (ESTs) derived from many different embryonic and adult tissues, suggesting that it may have a common role in different developmental contexts. One prototype member is the IFN-inducible human 9-27 (identical to the Leu-13 antigen) protein in leukocytes and endothelial cells, a cell surface component of a multimeric complex involved in homotypic adhesion and transduction of antiproliferative signals (Deblandre, G. A. et al. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 270, 23860-23866 (1995); Evans, S. S., Collea, R P., Leasure, J. A. & Lee, D. B. IPN-a induces homotypic adhesion and Leu-13 expression in human B lymphoid cells. J. Immunol. 150, 736-747 (1993); Evans, S. S., Lee, D. B., Han, T., Tomasi, T. B. & Evans, R. L. Monoclonal antibody to the interferoninducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells. Blood 76, 2583-2593 (1990)).
One prototype Fragilis family member, human 9-27 (identical to Leu-13 antigen), is inducible by interferon in leukocytes and endothelial cells, and is located at the cell surface as a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals (Deblandre, 1995).
The BLASTN search revealed that the Fragilis sequence was found in ESTs derived from many different tissues both from embryos and adults, indicating that Fragilis may play a common role in different developmental and cell biological contexts. Database searches reveal a sequence match with the rat interferon-inducible protein (sp:INIB RAT, pir:JC1241) with unknown function. The Fragilis sequence appears six times in our screen, indicating high level expression in PGcs.
The second gene, Stella, encodes a 150 amino acid protein, of 18 kD. The nucleic acid sequence of mouse Stella is set out in SEQ ID NO: 1.
It has no sequence homology with any known protein, contains several nuclear localisation consensus sequences and is highly basic pI (pI=9.67, the content of basic residues—23.3%), indicating a possible affinity to DNA. Furthermore a potential nuclear export signal was identified, indicating that Stella may shuttle between the nucleus and the cytoplasm. BLASTN analysis revealed that the Stella sequence was found only in the preimplantation embryo and germ line (newborn ovary, female 12.5 mesonephros and gonad etc.) ESTs indicating its predominant expression in totipotent and pluripotent cells. Interestingly, we found that Stella contains in its N terminus a modular domain which has some sequence similarity with the SAP motif. This motif is a putative DNA-binding domain involved in chromosomal organisation. There is also apparently a splicing factor motif-like structure in its C terminus. These findings suggest a possible involvement of Stella in chromosomal organization and RNA processing.
Example 4 Identification of PGCs by Screening for Fragilis and Stella ExpressionAlthough PGCs are identified in Example 2 by analysis of BMP4, TNAP, Hoxb1, and Oct4, no single one of these genes can be taken as a marker for the PGC state. However, both Fragilis and Stella may be used as such.
The expression of Fragilis is examined. Primer pairs are designed (5′: CTACTCCGTGAAGTCTAGG, 3′: AATGAGTGTFACACCTGCGTG) and the same PCR reaction as above is performed. Fragilis expression was detected in germ cell competent cells. The definitive PGCs were recruited from amongst this group of cells showing expression of Fragilis.
The boundary of Stella expression in particular is well-defined, and the expression is substantially limited to PGCs. Therefore, Stella is used as a positive marker for the selection of PGCs. Primer pairs are designed for amplifying the C terminal portion of Stella (5′: GCCATTCAGATGTCTCTGCAC, 3′: CTCACAGCTTGAGGCTTCTAA). The PCR amplification is performed using 0.5 μl of the cDNA solution obtained from PGCs in Example 1 as a template according to the following schedule: 95° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min for 20 cycles. Among 83 samples tested, only those taken from PGCs show expression of Stella. Hence, Stella is a positive marker for the PGC fate.
Antibodies against Fragilis and Stella can be similarly used to detect pluripotent cells. Preferably, antibodies against Fragilis are used to detect germ cell competent cells, and antibodies against Stella are used to detect PGCs.
Accordingly, both Fragilis and Stella are positive markers for the PGC fate which can be used to positively identify PGC.
Identification of PGC by ISH
The in vivo expression of the two genes is examined by in situ hybridisation. The expression of Fragilis starts very weakly in the entire epiblast at E6.0-E6.5 (PreStreak stage) and becomes strong in the few cell layers of the proximal rim of the epiblast. BMP4 that is expressed in the extraembryonic ectoderm is one signalling molecule that is important for the induction of germ cell competence and expression of Fragilis. Other signals, such as interferons are likely to be involved in the induction of Fragilis. The expression becomes more intense at the proximo-posterior end of the developing primitive streak at the Early/Mid Streak stage and becomes very strong at this position from Late Streak stage onward. The expression persists until Early Head Fold stage and eventually disappears gradually. No expression is detected in the migrating PGCs at E8.5.
The expression of Stella starts at the proximo-posterior end of the developing primitive streak at Mid/Late Streak stage and becomes gradually strong at the same position from the later stage onward. The expression is specific and individual single cells stained in a dotted manner can be seen in the region where PGCs are considered to start differentiating as a cluster of cells. At Late Bud/Early Head Fold stage, some cells considered to be migrating from the initial cluster are stained as well as cells in the cluster. At E8.5 and E9.5, a group of cells considered to be the migrating PGCs are very specifically stained.
From these results, it is concluded that Fragilis is a gene which is upregulated during the process of lineage specification and germ cell competence, and subsequently of PGCs, when Stella is turned on after Fragilis to fix the PGC fate.
Accordingly, expression of Fragilis may be detected in a method of detecting lineage specification, and/or pluripotency, such as germ cell competence. Similarly, expression of Stella may be detected to detect commitment to cell fate, for example, commitment to fate as a primordial germ cell.
Example 5 Expression of Fragilis and Stella During Germ Line DevelopmentAntibodies against Stella and Fragilis are used to detect expression of these genes in early embryos. It is found that each of these genes is expressed in primordial germ cells. In particular, we find that Fragilis is the first gene to mark PGC competent cells at the time of germ cell allocation. Stella is expressed only in the lineage-restricted founder PGCs and thereafter in the germ cell lineage.
Fragilis is expressed in pluripotent ES and EG cells. During the derivation of EG cells from PGCs, it is found that Fragilis expression re-appears on EG cells. Late PGCs are negative for Fragilis after specification of these cells is completed.
There is strong Fragilis expression at the base of incipient allantois where the founder PGC population differentiates in the E7.25 embryos. Fragilis expression persisted until E7.5, but it was not detected in migrating PGCs at E8.5. Fragilis is first detected in germ cell competent proximal epiblast cells. Fragilis expression can be induced in the epiblast cells when combined with the tissues extraembryonic ectoderm tissues, which is the source of BMP4. In the BMP4 mutant mice, there is no expression of Fragilis, consistent with the absence of PGCs in these embryos (Lawson et al., 1999).
Stella expression which is strong in PGCs is downregulated in EG cells. There is also low level expression of Stella in ES cells. Stella and Fragilis are detectable in ES and EG cells by Northern blot analysis. Stella is first detected at E7.0 in single cells within the distinctive cluster of lineage-restricted PGCs, and thereafter in migrating PGCs and subsequently when they enter the gonads.
Intracellular localisation of Stella and Fragilis is also determined. Fragilis localised to a single cytoplasmic spot at the Golgi apparatus, as well as in the plasma membrane. Stella comprises a putative nuclear localisation signal and nuclear export signal, and is localised in both the cytoplasm and nucleus.
Fragilis is observed in the Golgi apparatus as well as in the plasma membrane of PGCs. The cell surface localization of Fragilis is expected as a member of the interferon inducible gene family [Deblandre, 1995]. Expression of Fragilis in the proximal rim of the epiblast marks the onset of germ cell competence. Fragilis has an IFN response element upstream of its exon 1, so it is very likely to be induced by IFN after initial priming by BMP4 of the proximal epiblast cells. These IFN inducible proteins can from a multimeric complex with other proteins such as TAPA1, which is capable of transduction of antiproliferative signals, which may be why the cell cycle time in founder PGCs increases from 6 to 16 hr, while the somatic cells continue to divide rapidly.
Stella, which has the putative nuclear localization signal and a nuclear export signal, was observed in both the cytoplasm and the nucleus. The onset of Stella is followed by the loss of Fragilis expression by E8.5. Therefore, Fragilis expression marks the onset of germ cell competence and Stella expression marks the end of this specification process. Expression of Stella in the founder PGCs marks an escape from the somatic cell fate and consistent with their pluripotent state. These studies indicate that specific set of genes are required to impose a germ line fate on cells that may otherwise become somatic cells. Stella, with its potential to shuttle between the nucleus and cytoplasm, could have a role in transcriptional and translational regulation, since many organisms possess elaborate transcriptional mechanisms to prevent germ cells from becoming somatic cells. Expression of Stella in the oocyte and preimplantation embryos indicates that it has a wider role in totipotency and pluripotency.
Example 7 The Link Between Fragilis and StellaOnly some of the cells that express Fragilis, ended up showing expression of Stella Only those cells with the highest levels of Fragilis expression become PGCs and began to express Stella. Furthermore, Stella positive PGCs never show expression of Hoxb1. More importantly, only somatic cells with lower levels of Fragilis expression, show Hoxb1 expression. Furthermore, only the somatic cells show expression of two other homeobox-containing genes, Lim1 and Evx-1. Therefore lack of expression of Hoxb1, Evx-1 and Lim1, appears to be important for the specification of germ cell fate.
Our experiments also show that Oct4 is not a definitive marker of PGC, Previously, Oct4 expression is demonstrated in totipotent and pluripotent cells [Nichols, 199, Pesce, 1998; Yeom, 1996]. However, we find that Oct4 is expressed to the same extent in all PGCs and somatic cells. We do however find expression of T (Brachyuri) and Fgf 8 in PGCs indicating that PGCs are recruited from amongst embryonic cells that are initially destined to become mesodermal cells.
Example 8 PGC SpecificationThe founder PGCs and their somatic neighbours share common origin from the proximal epiblast cells. By analysing the founder PGC and the somatic neighbour, a systematic screen for critical genes for the specification of germ cell fate has been established. Fragilis is an interferon (IFN) inducible gene that can promote germ cell competence and homotypic association to demarcate putative germ cells from their somatic neighbours, and such an example may apply to other situation during development. Expression of Stella occurs in cells with high expression of Fragilis. Fragilis is no longer required once germ cell specification is complete, but Stella expression continues in the germ cell lineage. Stella may also be important throughout in the totipotent/pluripotent cells since it is also expressed in oocytes and early preimplantion development embryos.
Example 9 Germ Line and Pluripotent Stem CellsPGCs can be used to derive pluripotent embryonic germ (EG) cells. However, unlike EG cells, PGCs do not participate in development if introduced into blastocysts. They either cannot respond to signalling molecules, or that they are transcriptionally repressed. PGCs once specified do not express Fragilis on their cell surface. However, EG cells clearly show expression of Fragilis on their cell surface as do ES cells. Both EG and ES cells express Stella as judged by Northern analysis, although Stella is expressed at a lower level in ES and EG cells than in PGCs. Fragilis and Stella therefore have a role in pluripotent stem cells. These genes are therefore markers of these pluripotent stem cells, where they may also have a role in conferring pluripotency on these stem cells.
Example 10 Proposed Roles of Fragilis and Stella in PGC SpecificationFragilis as a typical IFN-inducible cell surface protein, probably shares certain properties common to all of these family members (Deblandre, G. A. et al. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 270, 23860-23866 (1995); Evans, S. S., Collea, R. P., Leasure, J. A. & Lee, D. B. IFN-a induces homotypic adhesion and Leu-13 expression in human B lymphoid cells. J. Immunol. 150, 736-747 (1993); Evans, S. S., Lee, D. B., Han, T., Tomasi, T. B. & Evans, R. L. Monoclonal antibody to the interferoninducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells. Blood 76, 2583-2593 (1990)).
The acute but transient expression of fragilis is itself consistent with the kinetics of IFN-inducible genes that can increase by up to 40-fold within 1 h, and decline quickly after IFN withdrawal (Friedman, R. L., Manly, S. P., McMahon, M, Kerr, I. M. & Stark, G. R. Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells. Cell 38, 745-755 (1984)). This Fragilis positive assembly of cells could correspond to about 100 TNAP positive cells (Lawson, K. A. & Hage, W. J. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found. Symp. 182, 68-84 (1994); Ginsburg, M., Snow, M. H. & McLaren, A. Primordial germ cells in the mouse embryo during gastrulation. Development 110, 521-528 (1990)), which is larger than the number of stella positive cells.
According to our estimates, the stella positive cluster in the 129/SvEv mouse strain consists of approximately 36-43 cells, which is close to the expected 45 nascent PGCs. The fragilis positive cells probably form a community of cells through homotypic adhesion (Evans, S. S., Collea, R. P., Leasure, J. A. & Lee, D. B. IFN-a induces homotypic adhesion and Leu-13 expression in human B lymphoid cells. J. Immunol. 150, 736-747 (1993); Evans, S. S., Lee, D. B., Han, T., Tomasi, T. B. & Evans, R. L. Monoclonal antibody to the interferoninducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells. Blood 76, 2583-2593 (1990)), from which the founder PGCs are recruited, thus demarcating them from most of the cells destined for somatic tissues. These IFN-inducible cell surface proteins are capable of transduction of antiproliferative signals (Deblandre, G. A. et al. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 270, 23860-23866 (1995)), which is a probable mechanism by which the cell cycle time in the nascent PGCs increases from 6 to 16 h, while the somatic cells continue to divide rapidly.
The induction of fragilis in epiblast cells may not by itself be sufficient for the expression of stella, as shown by our in vitro studies—induction may require a specific signal thought to be within the niche, for PGC specification in vivo (Lawson, K. A. et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 4214436 (1999); McLaren, A. Signaling for germ cells. Genes Dev. 13, 373-376 (1999)). This signal could be a specific ligand that binds to fragilis during the specification of germ cell fate. Once nascent PGCs are established, expression of fragilis is diminished by E8.0, thus freeing the PGCs from homotypic adhesion for their migration into the genital ridge (Wylie, C. Germ cells. Cell 96, 165174 (1999); Gomperts, M., Garcia-Castro, M., Wylie, C. & Heasman, J. Interactions between primordial germ cells play a role in their migration in mouse embryos. Development 120, 135-141 (1994)). fragilis must have other functions, as it is apparently expressed elsewhere in developing embryos. In this context, we also note fragilis expression in pluripotent ES and embryonic germ cells (data not shown), where it may have a role in the propagation of the pluripotent state.
The role of stella may in part be regulated by its potential to shuttle between the nucleus and cytoplasm. We have observed, for example, that overexpression of stella in somatic cells causes the protein to be retained in the cytoplasm and not in the nucleus, as is predominantly the case in PGCs (data not shown). A particularly critical event involved in the specification of PGCs is repression of the region-specific homeobox genes, by which nascent PGCs escape from the somatic cell fate. As the expression of stella is most intimately connected with the generation of PGCs, this gene is a chief candidate for either initiating or maintaining repression of Hox genes in PGCs. The detection of stella in the oocyte and through pre-implantation development (B. Payer et al., unpublished data; Sato, M. et al. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech. Dev. 113, 91-94 (2002)) suggests that it may serve a critical role during all the phases of totipotent/pluripotent states in mice.
Example 11 Fragilis 2, Fragilis 3, Fragilis 4 and Fragilis 5Specification of primordial germ cells in mice depends on instructive signalling events, which act first to confer germ cell competence on epiblast cells, and second, to impose a germ cell fate upon competent precursors. fragilis, an interferon-inducible gene coding for a transmembrane protein, is the first gene to be implicated in the acquisition of germ cell competence.
In this and the following Examples (Examples 11 to 20), we describe four additional fragilis-related genes, fragilis2-5, which are clustered within a 70 kb region in the vicinity of the fragilis locus on Chr 7. These genes exist in a number of mammalian species, which in the human are also clustered on the syntenic region on Chr 11. In the mouse, fragilis2 and fragilis3, which are proximate to fragilis, exhibit expression that overlaps with the latter in the region of specification of primordial germ cells. Using single cell analysis, we confirm that all these three fragilis-related genes are predominant in nascent primordial germ cells, as well as in gonadal germ cells.
The Fragilis family of interferon-inducible genes is tightly associated with germ cell specification in mice. Furthermore, its evolutionary conservation suggests that it probably plays a critical role in all mammals. Detailed analysis of these genes may also elucidate the role of interferons as signalling molecular during development.
Example 12 Background to ExamplesGerm line determination in the mouse is thought to occur through instructive signalling in the gastrulating post-implantation embryo [1,2]. First, proximal epiblast cells acquire germ cell competence at E6.5, partly in response to extraembryonic ectoderm-derived signalling molecules. A subset of these competent cells then acquire a primordial germ cell (PGC) fate and a population of approximately 45 founder germ cells are detected in the posterior proximal region of the embryo at the base of the incipient allantoic bud on E 7.5 [1, 2]. The secreted signalling molecules, BMP4, BMP8b and BMP2 as well as components of the BMP signal transduction pathway, including Smad1 and Smad5, appear to be involved in the specification of PGCs [3-7]. However, in vitro culture studies and analysis of BMP4-deficient mice suggest that an additional signal may also be required for the acquisition of PGC fate, but its identity is yet unknown [2,3].
We have identified fragilis, a putative interferon-inducible gene, which codes for a transmembrane protein that is apparently associated with the acquisition of germ cell competence by epiblast cells [8]. Extraembryonic ectoderm is able to induce fragilis expression in epiblast tissue, and BMP4 is required for this induction [8]. fragilis is expressed in proximal epiblast at E6.5, the region in which PGC-competent cells reside according to clonal analysis [1]. As these proximal cells move to the posterior proximal region during gastrulation, fragilis expression increases within a community of cells at the base of the incipient allantoic bud. Cells with the highest expression of fragilis initiate the germ cell-characteristic expression of TNAP and stella/PGC-7 [8, 9, 10]. These nascent PGCs with high expression of fragilis also show repression of Hox genes, including Hoxb1 in nascent PGCs [8].
In view of the strong association of fragilis with PGC specification, we have started to investigate further how this gene may be regulated and what precise function it serves during germ cell development. Towards this objective, we now report that fragilis belongs to a novel murine gene family, comprising five members, which code for five highly similar transmembrane proteins. More importantly, the genes are clustered within a 70 kb genomic region. As we found several homologues of the Fragilis family in human, cow and rat, they seem to be evolutionarily conserved amongst mammalian species. Most if not all homologous genes have been reported to be responsive to interferon signalling, which is in agreement with the presence of conserved interferon stimulable response elements (ISREs) within at least the murine and human loci. Furthermore, our in situ hybridisation and single cell expression analysis reveal that the two members located close to fragilis, fragilis2 and fragilis3, are also expressed in nascent PGCs, although their overall expression pattern in post-implantation embryos in other respects is distinct. Studies on the Fragilis family of genes could therefore be crucial for our understanding of PGC specification, especially since their homologues have been implicated in mediating homotypic cell adhesion and lengthening of the cell cycle time [14, 15]. These studies may also show how interferons act as signalling molecules, which has hitherto not been considered in the context of embryonic development.
Example 13 Materials and Methods: Database Searches and AnimalsEnsembl and NCBI genome browsers are used for data retrieval.
Embryos and genital ridges used for in situ hybridisation experiments came from 129×129 or F1xGoF1 mothers, respectively. Embryos and genital ridges used for single cell analysis came from 129×SvEv or Oct4GFP(129)×MF1 mothers, respectively. The day of the vaginal plug was designated as E0.5. Embryos were staged according to Downs and Davies [22].
Example 14 Materials and Methods: In Situ Hybridisation3′-fragments of fragilis and fragilis2-5 cDNAs were PCR amplified using the primers described below, and cloned into pGEMT vector (Promega). DIG-labelled antisense RNA probes were synthesized using DIG RNA labelling kit (Sp6/T7; Roche). In situ hybridisation on embryos and urogenital ridges was performed as described [23, 24]. Hybridisation was carried out using 1 μg/ml DIG-labelled RNA probe in hybridisation buffer (50% formamide, 1.3×SSC (pH 5), 5 mM EDTA (pH 8), 50 μg/ml yeast RNA, 0.2% Tween-20, 0.5% CHAPS, 100 μg/ml heparin in DEPC treated H2O) at 70° C. over night. Hybridised probe was detected using alkaline phosphatase conjugated anti-DIG Fab fragments (Roche) and BM Purple alkaline phosphatase substrate (Roche).
Example 15 Materials and Methods: Preparation, PCR and Southern Blot Analysis of Single Cell cDNAsEarly bud stage embryos (E 7.5) and genital ridges (E 11.5) were isolated in DMEM/10% fetal calf serum/25 mM HEPES (pH 7.4). Fragments bearing primordial and gonadal germ cells, respectively, were dissected out and dissociated into single cells. The latter were picked using mouth pipettes and their cDNAs were amplified as described previously [25]. The following primers were used in order to PCR amplify stella cDNA and 3′-fragments of fragilis and fragilis2-5 cDNAs (25 cycles of amplification):
PCR products were run on 1.5% agarose/TBE electrophoresis gels. For Southernblot analysis, single cell cDNAs were blotted onto Hybond-N+ membranes (Amersham) and probed with 32αP dCTP-labelled DNA probes comprising the 3′ regions of fragilis, fragilis2 and fragilis3 cDNAs and full length stella cDNA. GAPDH was used as loading control. Blotting signal was detected using a Fuji film FLA 5000 scanner. Signal strength was quantified in relation to GAPDH signal, whereby relative gene expression was calculated as ratio of gene signal to GAPDH signal and this ratio was subsequently normalized by division through the highest hybridisation signal per blot. For dotblot analysis, full length fragilis cDNAs were blotted and probed with 32αp dCTP-labelled 3′ probes.
Using the cDNA sequence of fragilis as a template to search the ensembl genome browser (www.ensembl.org), we identified eight mouse genes with moderate to high DNA sequence similarity to fragilis (45-74%). ESTs from a variety of embryonic and adult tissues have been reported for five of these genes, of which four possess a two-exon structure similar to fragilis. Analysis of the genomic location of the latter revealed that the four genes cluster around the fragilis locus within a 70 kb region on the distal tip of mouse Chr 7 (F5). We therefore named the four novel genes fragilis2-5, reflecting their genomic location, similarity to fragilis and germ cell associated expression pattern (see below;
To determine whether the Fragilis genes are evolutionary conserved, we have identified four homologues of mouse Fragilis in the human genome on Chr 11 (p 15.5), a region which is indeed syntenic to the Fragilis family locus on mouse Chr 7 (
The murine family of fragilis and related genes code for five highly similar transcripts of 104 to 144 amino acids, each containing two predicted transmembrane domains (
We analysed the expression pattern of the five Fragilis family genes by whole mount in situ hybridisation using probes that span the 3′ region (150-200 bp) of the corresponding mRNAs. These probes show no significant cross-hybridization between members of the Fragilis family as judged by dotblot analysis (data not shown). As reported, we saw expression of fragilis restricted to the epiblast at E5.5 and E6.5. More importantly, around E7.5, expression of fragilis is intense within a population of cells at the base of the allantois in the region where PGC specification occurs (
At E8.5, fragilis expression is seen in cells at the base and within the proximal third of the allantois (
In contrast to the first three members of the family, neither fragilis4 nor fragilis5 showed expression at early post-implantation stages (E7.0-E8.5, data not shown). Consequently, only the three genes at the centre of the family cluster, that is fragilis, fragilis2 and fragilis3 are expressed in the embryo between E5.5 and E9.5. While their expression pattern is distinct, there is a striking overlap within the region where founder germ cells are located. This suggests that the three neighbouring genes, fragilis, fragilis2 and fragilis3, may share regulatory elements that are likely to be present within the cluster. These regulatory elements may also be responsible for the genes' overlapping expression pattern specifically around the region of nascent PGCs.
Example 18 Single Cell Analysis of Fragilis, Fragilis2 and Fragilis3 in PGCs and Somatic NeighboursTo obtain more precise information on the expression of the new Fragilis family members in the context of germ cell specification, we tested single cell cDNAs from PGCs and surrounding somatic cells sited at the base of the incipient allantoic bud in E7.5 embryos. Both fragilis2 and fragilis3 were expressed in nascent PGCs, which show transcription of the germ cell marker stella/PGC7 (
During the developmental stages directly subsequent to PGC specification, all three Fragilis family genes are expressed in a population of cells associated with the allantois and in a location where premigrating PGCs are thought to reside (
In this study we describe the identification of the murine Fragilis gene family, which appears to be conserved amongst mammalian species, and whose members code for five highly similar transmembrane proteins. Three members of the Fragilis family, fragilis, fragilis2 and fragilis3, exhibit expression, which is associated with germ cell specification and development Located at the cell membrane, the Fragilis proteins may be crucial for mediating interactions amongst germ cells and their surrounding neighbours. While the three genes are expressed earlier at E5.5 and thereafter to a varying extent, they all show upregulation of expression within nascent PGCs. It is likely that a cis control element exists within the locus that is required for this expression, which continues within gonadal PGCs. Future studies will elucidate where these control elements are located and how they regulate expression of the fragilis-related genes.
Although the five Fragilis family members are clustered within a small genomic region, it appears that neither fragilis4 or fragilis5 show expression in early embryos or embryonic germ cells. It is striking that these two members are located at the periphery of the cluster in contrast to the centrally located fragilis, fragilis2 and fragilis3 genes. This lack of expression may be due to the presence of boundary elements, which might restrict the action of control elements to genes present within the centre of the cluster. Since sequence comparison suggests that gene duplications may have occurred independently in the two species, it appears that a certain evolutionary constrain may exist on duplication and maintenance of the duplicated genes within immediate neighborhood. Since the four human homologues of the Fragilis family in the syntenic region are also arranged in a genomic cluster and are highly similar to the family genes, it is tempting to suggest that they may also serve similar functions as in the mouse.
The presence of several interferon stimulable response element (ISRE) consensus sequences within the Fragilis locus, together with the similarity of the genes to their interferon-inducible human and bovine counterparts, suggest very strongly that fragilis and the fragilis-related genes are responsive to interferons. Indeed, the ISRE tandem repeat present in the 5′ flanking region of human Ifitm1, Ifitm2 and Ifitm3 genes is also present in the 5′ flanking region of fragilis exon 1 [11]. Interferons, as secreted signalling molecules, have so far been implicated mainly in the process of immune response, the inhibition of cellular growth and the control of apoptosis [19]. Although interferons are expressed in the post-implantation embryo, their role during development has not been addressed in detail [20, 21]. Our studies have pointed to a possible involvement of interferons in germ cell development. Future work will determine whether the Fragilis genes respond to interferon signals in all or some instances where the genes are expressed, which we expect in view of the presence of conserved ISRE elements in the mouse and human loci.
Example 20 ConclusionWe have identified the Fragilis family of interferon inducible genes, which code for transmembrane proteins. The five members are arranged in a cluster within a genomic region of 70 kb in the mouse that also contains ISRE elements. The centrally located fragilis, fragilis2 and fragilis3 genes are of particular interest, because they are expressed in the region where germ cell specification occurs. The family is evolutionary conserved amongst mammalian species where it may serve similar functions. Detailed studies of the Fragilis family may also show what role interferons have in embryonic development.
Example 21 Stella is a Maternal Effect Gene Required for Normal Early Development in MiceIn this and the following Examples (Examples 21 to 25), we have investigated the effects of a targeted mutation of stella in mice. Maternal inheritance in mammalian oocytes includes proteins important for totipotency and epigenetic modifications1, as well as factors crucial for early development, which are transcribed from so called maternal effect genes2-7.
Amongst these maternally inherited proteins is Stella, which is also expressed in preimplantation embryos, primordial germ cells, and pluripotent cells8,9. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella-null offspring, stella-deficient females showed severely reduced fertility, which is due to a lack of maternally inherited Stella in their oocytes.
Stella is a maternal effect gene, as the phenotypic effect on embryonic development is a consequence of the maternal stella mutant genotype. Indeed, we demonstrate that embryos lacking Stella-protein are compromised in preimplantation development and rarely reach the blastocyst stage. Furthermore, we show that STELLA that is expressed in human oocytes10 is also expressed in human pluripotent cells and in germ cell tumours. Interestingly, human chromosome 12p, which harbours STELLA is consistently overrepresented in these tumours11. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumours.
The aim of this study was to determine the role of stella by loss of function analysis in mice. In our previous work, we have shown that expression of stella (also called PGC7) is activated during the process of germ cell specification at E7.25 specifically in the founder population of lineage restricted primordial germ cells (PGCs)8,9. Thereafter it is expressed in the germ line until about E15.5 in male and E13.5 in female gonads. Expression of stella resumes in the immature oocytes in newborn ovaries, and it is subsequently detected in maturing oocytes and in preimplantation embryos (
Immunofluorescence
Embryos were fixed in 4% paraformaldehyde for 15 minutes, washed 3 times with PBS and permeabilised in AB-buffer (1% Triton-X100, 0.2% SDS, 10 mg/ml BSA in PBS), which was also used for the following antibody incubations and washes. They were then incubated in primary antibody (anti-Stella9 1:200, anti-PGC78 1:2000) overnight at 4° C., washed 3 times and incubated with secondary antibody for 1-2 hours at room-temperature (Alexa 564, Molecular probes, 1:500). After 3 further washes in AB-buffer, embryos were rinsed once in PBS and incubated at 37° C. with 0.1 mg/ml Rnase A (Roche) in PBS for 30 minutes. Finally embryos were incubated for 10 minutes in PBS with propidium iodide (2 μg/ml) and mounted on slides in Vectashield (Vector Laboratories) mounting medium, which also contained propidium iodide.
For E11.5 PGC-stainings, genital ridges were washed in PBS, treated for 10 minutes at 37° C. with Trypsin/EDTA (Gibco), diluted in PBS and dissociated into a cell suspension. Cells were allowed to settle down on poly-L-lysine treated slides and fixed with 3% formaldehyde for 15 minutes. After permeabilisation with 0.2% Triton X-100 in PBS and 3 washes in PBS cells were blocked with 3% BSA in PBS (also used for subsequent washes and antibody dilutions) for 40 minutes and incubated with primary antibodies (anti-Stella 1:100, anti-SSEA1 (=TG1), P. Beverley 1:2) overnight at 4° C. Then the cells were washed and incubated with secondary antibodies (Alexa 564, Alexa 488, Molecular probes, 1:500) for 1.5 hours. After washing, Rnase (0.1 mg/ml) treatment was done for 1 hour at room temperature and the cells were mounted with Vectashield containing Toto-3 (Molecular probes, 1:1000).
Immunofluorescence was visualized on a BioRad Radiance 2000 confocal microscope.
Identification of Stella-Homologues
Human STELLA was identified by blasting the mouse Stella protein sequence against the translated human genome sequence using the Ensembl server (http://www.ensembl.org). The only hit showing the same intron-exon structure as the mouse gene is located on the syntenic region (
The putative rat-stella sequence was mapped as above and deduced from the alignment of the mouse cDNA sequence with the syntenic rat genome sequence.
RT-PCR Analysis of Human Tissues
1 μg total RNA of each human tissue (source: Ambion and see acknowledgements) was reverse transcribed into 1st strand cDNA with Superscript II reverse transcriptase (Gibco) for 1 hour at 37° C. 1 μl of this cDNA was amplified by a 30 cycle PCR-reaction using primers for human STELLA (5′-CAATTTGAGGCTCTGTCATCAG-3′,5′-TTCATCTCACTGACTTTGGGC-3′) or ribosomal protein L32 (5′-AGTTCCTGGTCCACAACGTC-3′,5′-TGCACATGAGCTGCCTACTC-3′).
ES-Cell Manipulation and Knockout Verification
The targeting construct consisted of 1.5 kb of upstream and 4.1 kb of downstream genomic sequence flanking the second exon of stella. The 5′ arm terminated after the first 32 bp of exon 2, which was fused to an IRES lacZ reporter, followed by a promoted neo selectable marker. The construct was linearized and electroporated into CCB mouse embryonic stem (ES) cells which were placed under selection. Individual G418-resistant clones were picked and screened for correct integration of the targeting construct by PCR using a vector primer and a primer external to the 5′ arm. 288 clones were screened of which two exhibited the expected size bands in the PCR. Homologous recombination was also confirmed by Southern blot using 5′, 3′ and neo-probes on NcoI and EcoRI digested genomic DNA. The correctly targeted ES-cell clone F4 was injected into MF1 and C57BL/6 blastocycsts to produce chimeric mice. Germline transmission was achieved by breeding the male chimeras with 129Sv/Ev females. All analysis was done on the inbred 129Sv/Ev background. To confirm that the stella gene was correctly inactivated, mice were genotyped by Southern blot as above (
Counting of Primordial Germ Cells
Embryos of stella-heterozygous intercrosses were dissected out at E8.5, fixed with 4% paraformaldehyde and stained for TNAP-positive PGCs with α-naphthyl phosphate/fast red TR solution (Sigma) as previously described20,26. The posterior parts of the embryos were flattened under coverslips and used for counting PGCs, while the anterior parts were used for genotyping by PCR.
Histology
Testes and ovaries from adult mice were fixed in Bouin's fixative at 4° C. overnight and washed thoroughly in 80% ethanol. After dehydration through an ethanol series they were transferred into xylene and embedded in Paraplast Plus wax (Sigma). 8 μm sections were cut, rehydrated and stained with Ehrlich's Haematoxilin (BDH) and 1% eosin (Sigma) After dehydration, slides were mounted with DPX (BDH).
Matings and In Vitro Culture
All studies for the assessment of fertility and embryonic development were done using natural matings. Mice were kept on a constant light/dark cycle and mating was assumed to have happened in the middle of the dark period before a vaginal plug was detected (E0.5=midday on day of plug). Embryos were collected by flushing oviducts/uteri at the time of the observed stages (E0.5-E3.5) or at E1.5, if they were cultured. Culturing was done under 5% CO2 in KSOM medium.
Work on animals was performed under Home Office project licenses PPL80/1280 and PPL80/1706.
Generation of Stella-GFP Mice
Using the stella-cDNA as a probe, we screened a gridded genomic 129 pBeloBAC library (Genome Systems St Louis, Mo.) to identify a clone harbouring the stella locus. We subcloned 11.5 kb of genomic sequence including about 8.5 kb upstream sequence and exon 1, intron 1 and the start of exon 2 and fused it in frame to eGFP (Clontech) and a SV40-polyadenylation signal. This sequence was then injected into pronuclei of B6CBA F2 zygotes, to generate transgenic mice. The transgene was maintained on the same genetic background and the onset of expression of the paternal allele was observed by mating stella-GFP transgenic males with non-transgenic females.
The cDNAs of the Stella homologues mentioned in this study have the following GenBank accession numbers: mouse Stella (AY082485), rat Stella (BK001414, pending), human STELLA (AY317075, pending).
Example 23 Stella HomologuesWe have now identified stella homologues in the rat and human genomes, which show the same exon-intron structure, and are located within the syntenic chromosomal regions (see
To study the expression of human STELLA, we performed RT-PCR analysis on pluripotent cell lines and reproductive organs (
To begin to address functions of stella, we generated stella knockout (stella) mice (
As stella is detected in the founder PGCs, we examined stella−/− mice for any effects on development of germ cells. Examination of germ cells at E8.5 in mutant embryos by tissue non specific alkaline phosphatase (TNAP) activity, a marker of PGCs16, revealed no significant differences in the numbers of PGCs compared to those in wild-type embryos (
Next, we examined if development progressed normally from oocytes of stella−/− females that lack maternal inheritance of Stella. Despite the ovulation of normal numbers of Stella-deficient oocytes, female stella-mice displayed a strongly reduced fertility. When stella−/− females were mated with wild-type males, only a low percentage of matings (detected by vaginal plugs) (24%,
Next we wanted to know, if the Stella protein in preimplantation embryos (
We then addressed the question concerning the embryonic stages at which the absence of Stella affects development. As we have so far not obtained any live young from matings between stella−/− males and stella−/− females, we examined embryos from these matings, and compared it with embryos from matings between wild-type or stella−/− males with wild-type or stella+/− females (
In conclusion, we demonstrate that the maternal inheritance of Stella is needed for normal embryonic development. Depletion of Stella from the oocytes compromises this process, resulting in a progressive decline in the numbers of blastocysts, fewer implants and a poor yield of viable young. Stella is a basic protein with a SAP-like domain25 and a splicing factor-like motif and therefore likely to have a role in chromosomal organisation or RNA metabolism. We propose to look for the interacting partners and the biochemical activity of the conserved domains of Stella to elucidate its role in early development. Despite a lack of gross abnormalities in germ cell development in stella−/− mice, we cannot rule out subtle effects. One possibility is functional redundancy through compensation by stella-related genes. There are several stella-like sequences in the mouse genome, although these are likely to be pseudogenes (data not shown). STELLA is also expressed in human oocytes10, where it is likely to play a similar role in early development as in mice. As the highest expression of STELLA is in a human testicular germ cell tumour, this could serve as a diagnostic marker or be of therapeutic value in the future. The conservation of the syntenic chromosomal region harbouring STELLA, together with NANOG and GDF3 on chromosome 12p is noteworthy as it is associated with pluripotency, teratocarcinomas and germ cell tumours in humans. The role of likely coordinated regulation of all key genes within the region may provide evolutionary insights into aspects of germ cell development and germ cell tumours, as well as on pluripotency and maternal effect genes.
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Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents (“application cited documents”) and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.
Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.
Claims
1-17. (canceled)
18. A polypeptide comprising a human Stella amino acid sequence as shown in SEQ ID NO:4, or a sequence that is at least 95% identical thereto, wherein the polypeptide can serve as a marker of pluripotency.
19. A polynucleotide sequence that codes for the polypeptide of claim 1, suitable for use as a marker of pluripotency.
20. The polynucleotide sequence of claim 19, wherein the sequence is as shown in SEQ ID NO:3 or a sequence that is at least 95% identical thereto.
21. A method for identifying a pluripotent cell comprising detecting expression of a Stella nucleic acid sequence in the cell.
22. The method of claim 21, wherein the nucleic acid sequence is as shown in SEQ ID NO:3 or a sequence that is at least 95% identical thereto.
23. A method for identifying a pluripotent cell comprising detecting expression of a Stella polypeptide in the cell.
24. The method of claim 23, wherein the Stella polypeptide is as shown in SEQ ID NO:4, or a sequence that is at least 95% identical thereto.
25. A polynucleotide expression construct suitable for detecting pluripotency in a cell, comprising: wherein expression of the coding sequence of the reporter gene is in operable combination with and under the control of the transcriptional control sequences of the human Stella gene, and wherein induction of expression of the reporter gene in the cell can be detected and thereby correlated with a pluripotent phenotype in the cell.
- i) at least the upstream sequences of a human Stella gene; and
- ii) the coding sequence of a reporter gene,
26. The polynucleotide expression construct of claim 25, wherein the upstream sequences of the Stella gene include the 5′ untranslated region of the Stella gene.
27. The polynucleotide expression construct of claim 25, wherein the upstream sequences of the human Stella gene comprise up to about 8.5 kb of the nucleic acid sequence located upstream of the first exon of the human Stella coding sequence.
28. The polynucleotide expression construct of claim 25, wherein the expression construct is in the form of a transgene that can be integrated into the genome of the cell.
29. The polynucleotide expression construct of claim 25, wherein the cell is a stem cell.
30. The polynucleotide expression construct of claim 25, wherein the cell is selected from one of the group consisting of: a pluripotent cell; an embryonic stem cell; an embryonic germ cell; a germ cell tumour cell; and a primordial germ cell.
31. The polynucleotide expression construct of claim 25, wherein the cell is a human cell.
31. The polynucleotide expression construct of claim 25, wherein the reporter gene is a green fluorescent protein (GFP) gene.
32. A cell comprising a polynucleotide expression construct of claim 25.
33. A non-human transgenic animal comprising a cell that comprises a polynucleotide expression construct of claim 25.
Type: Application
Filed: Jan 20, 2009
Publication Date: Aug 20, 2009
Inventors: Mitinori Saitou (Cambridge), Azim Surani (Cambridge)
Application Number: 12/356,351
International Classification: A01K 67/033 (20060101); C07K 14/00 (20060101); C07H 21/00 (20060101); C12Q 1/68 (20060101); C12Q 1/02 (20060101); C12N 5/00 (20060101);
