Abstract: The present invention provides a method for producing cloned cows by employing in vitro maturation of oocytes and nuclear transfer techniques. The method comprises collecting donor somatic cell lines from cows; maturing oocytes extracted from an ovary in vitro; enucleating the oocyte by cutting a portion of the zona pellucida and squeezing out a portion of the cytoplasm, including the first polar body; inserting the donor somatic cell into the oocyte; electrofusing the cells to produce embryos; post-activating the embryos; transferring them into surrogate cows to produce cloned calves.

Abstract: The invention features methods of producing a mammal, e.g., a cloned or transgenic mammal. The method includes maintaining a reconstructed embryo in culture until the embryo is in the 2 to 8 cell stage of embryogenesis, transferring the embryo at the 2 to 8 cell stage into a recipient mammal, and allowing the embryo to develop into a mammal.

Abstract: Transgenic mice that produce high levels of humanized antibodies are described. Targeted gene replacement exchanges constant regions of the mouse immunoglobulin heavy and light chain genes with human genes, either through conventional gene targeting, or by use of the bacteriophage-derived Cre-loxP recombination system. The transgenic animals undergo antibody affinity maturation, and a class switch from the native immunoglobulin to the humanized form.

Abstract: The invention relates to methods of introducing a heterologous DNA sequence into a mouse embryonic stem cell wherein the DNA sequence is inserted by homologous recombination into a villin gene/I-SceI hybrid by creating a double strand break with I-SceI meganuclease. Subsequently, the mouse embryonic stem cells can be used to generate a transgenic mouse comprising the heterologous DNA sequence. Additionally, the methods can be used for gene replacement in ovo where a mouse oocyte containing a villin gene/I-SceI hybrid within its genome exists or is first generated. More generally, the methods can be used for the targeted insertion of a heterologous DNA sequence into any cell containing a villin gene/I-SceI hybrid sequence within its genome.

Abstract: An optimum time has been discovered for obtaining porcine oocytes with improved developmental competence. Harvested oocytes are matured by culturing in vitro, and then activated ˜42-46 hours after beginning of the culture period. Alternatively, ovulation is induced in a donor female using a gonadotrophic hormone (optionally monitored by ultrasonography), maturation is allowed to proceed in vivo, and then the harvested oocyte is activated ˜44-48 hours after inducing ovulation. Outside the optimal time frame, the ability of the oocytes to undergo parthenogenetic activation declines markedly. These methods overcome problems encountered with previous protocols for nuclear transfer and assisted reproduction in the pig.

Abstract: The present invention pertains to a method for treating obesity in a mammal which comprises reducing the biological activity of HMGI genes in the mammal. In another embodiment, the invention pertains to a method for treating a tumor in a patient by reducing the biological activity of normal HMGI genes which comprises administering to the patient a therapeutically effective amount of an inhibitor compound active against normal HMGI-C or HMGI(Y) genes. In another embodiment, the invention pertains to a method of producing a transgenic non-human mammal, the germ cells and somatic cells of which contain an inactivated HMGI gene sequence introduced into the mammal at an embryonic stage. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI proteins. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI genes.

Abstract: A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Abstract: A method of altering the phenotype of a bird comprises introducing a DNA sequence into somatic cells of a bird contained within an egg during in ovo incubation. The DNA sequence is selected to be effective to cause a change in phenotype, such as an increase in growth rate, feed efficiency, or both in the bird after hatch. A DNA sequence may further be selected to increase disease resistance or induce disease prevention by the expression of an antigen over a period of time.

Abstract: A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

Abstract: Transgenic mice are constructed by binding the “hHB-EGF/DTR” gene to the downstream of an albumin enhancer/promoter that is expressed specifically in hepatic parenchymal cells and introducing this unit into mice. After the “hHB-EGF/DTR” gene has been confirmed to be expressed specifically in hapatic cells, diphtehria toxin is administered to the transgenic mice to examine whether the hepatic parenchymal cells are disrupted. The hepatic cells of the transgenic mice can be selectively dirupted depending on the administration period of the diphtheria toxin.

Abstract: A method of producing a protein that degrades or detoxifies organic material is described. This method involves producing a non-human transgenic animal that produces such protein in its urine, and has stably integrated into its genome an exogenous gene encoding a protein that is detectable in the urine. Thus, a non-human transgenic animal that produced such protein in its urine, and a method of degrading or detoxifying organic materials also is described. Also a facility comprising a non-human transgenic animal that produce in its urine a protein that degrades or detoxifies organic material and a structure containing such animal is described. A method of altering a substance naturally found in urine is described. A DNA construct used in producing the non-human transgenic animal also is described.

Abstract: The present invention concerns products and methods particularly useful for activating and analyzing non-dividing cell nuclei. The featured products include activating egg extracts, cytostatic factor (CSF) extracts, kits containing these extracts, and a microchamber microscope slide useful in analyzing nucleus activation.

Abstract: The present invention provides for genetically engineered non-human animals, including but not limited to mice, which lack one or more endogenous neurofilament gene. Human neurofilament genes may be introduced into such genetically non-human animals (referred to hereafter as “knockout animals”) to produce improved models of the physiology of human neurofilament proteins which may be used to study human neurofilament-associated neurodegenerative conditions.

Abstract: A method comprising: a) altering a chromosomal sequence of a donor nucleus of a donor cell by introducing a pair of single-stranded targeting polynucleotides, and a recombinase into said donor nucleus of said donor cell, wherein said pair of targeting polynucleotides are substantially complementary to each other and each comprising a homology clamp that substantially corresponds to or is substantially complementary to a predetermined DNA sequence of said nucleus; and, b) transplanting said nucleus into an oocyte to produce a recombinant zygote.

Abstract: The present invention relates to a method for transfection of avian primordial germ cells which comprises a step of electroporating a foreign gene into avian primordial germ cells after addition of dimethylsulfoxide( “DMSO”) to culture medium containing the cells, and a method for production of transgenic Aves by employing the avian primordial germ cells transfected thereby. The DMSO-electroporation method of the invention has various advantages over the conventional transfection methods in light of high transfection efficiency, safety, and simple manipulation. Accordingly, a large number of primordial germ cells transfected with a gene of interest can be efficiently produced by the invention, which, in turn, may increase the rate of transfer of exogenous DNA into recipient embryos.

Abstract: Methods and cell lines for cloning ungulate embryos and offspring, in particular bovines and porcines, are provided. The resultant fetuses, embryos or offspring are especially useful for the expression of desired heterologous DNAs, and may be used as a source of cells or tissue for transplantation therapy for the treatment of diseases such as Parkinson's disease.

Abstract: The present invention relates to cloning technologies. The invention relates in part to immortalized and totipotent cells useful for cloning animals, the embryos produced from these cells using nuclear transfer techniques, animals that arise from these cells and embryos, and materials, methods, and processes for establishing such cells, embryos, and animals.

Abstract: A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Abstract: The present invention relates to materials and methods for cloning porcine animals. The invention relates in part to totipotent cells useful for cloning porcine animals, porcine embryos produced from such cells by employing nuclear transfer techniques, and porcine animals that arise from such cells and embryos.

Abstract: An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Abstract: Animals are produced following injection of adult somatic cell nuclei into enucleated oocytes. The invention provides a method for cloning an animal by directly inserting at least a portion of the adult somatic nucleus (including the minimum chromosomal material able to support development) into a recipient enucleated oocyte. Preferably, the nucleus is inserted by microinjection and, more preferably, by piezo electrically-actuated microinjection. The oocyte is activated prior to, during, or up to about 6 hours after insertion of the nucleus, by electroactivation or exposure to a chemical activating agent, such as Sr2+. The activated renucleated oocyte is allowed to develop into an embryo and is transplanted to a host surrogate mother to develop into a live offspring.

Abstract: Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Abstract: Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods. In particular, satellite artificial chromosomes that, except for inserted heterologous DNA, are substantially composed of heterochromatin are provided. Methods for use of the artificial chromosomes, including for gene therapy, production of gene products and production of transgenic plants and animals are also provided.

Abstract: The present invention relates to materials and methods for cloning porcine animals. The invention relates in part to totipotent cells useful for cloning porcine animals, porcine embryos produced from such cells by employing nuclear transfer techniques, and porcine animals that arise from such cells and embryos.

Abstract: A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Abstract: The present invention relates to recombinatorial substrates which include a promoter, a terminator, a gene positioned 3′ to the terminator and whose expression is to be controlled, and recombination sites on each side of the terminator such that when the substrate is treated with a specific recombinase the gene will be expressed. Recombinatorial substrates which have a promoter, a gene to be controlled, and recombination sites on each side of the gene which when treated with recombinase delete the gene are also provided. Also enclosed are methods of creating transgenic mammals carrying the recombinatorial substrate and methods for activating the recombinatorial substrate.

Abstract: An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Abstract: An improved method of nuclear transfer involving the transplantation of donor differentiated pig cell nuclei into enucleated pig oocytes is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring. Production of genetically engineered or transgenic pig embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Abstract: Methods and cell lines for cloning bovine embryos and offspring are provided. The resultant embryos or offspring are especially useful for the expression of desired heterologous DNAs.

Abstract: Provided is a method of parthenogenetically activating an unfertilized mammalian oocyte, comprising contacting an unfertilized mammalian oocyte with an oocyte-modifying agent followed by a reducing agent, wherein the mammalian oocyte is in contact with the oocyte-modifying agent and reducing agent, respectively, for a time and under conditions such that the unfertilized mammalian oocyte is activated. Also provided are methods for transplanting a nucleus from a cultured mammalian cell, mammalian embryo, mammalian fetus, or an adult mammal to a recipient mammalian oocyte; producing a cloned mammalian embryo; and producing a cloned mammal, as well as the cloned products produced by these methods. Finally, a method of studying genetic imprinting in mammals is also provided.

Abstract: Transgenic mice are described which serve as a model for Hepatitis C infection. The transgenic mice contain, in their germline and somatic cells, the Hepatitis C viral fragment CN2, N24 or CR under the control of a Cre-loxP switch-expression system. Administration of Cre to the mice results in a phenotype of increased serum GTP levels, emergence of acidophilic bodies in the liver, exfoliation of hepatic cells, hypertrophy and hyperplasia of Kupffer's cells, and conglomeration of lymphocytes.

Abstract: A method of reconstituting a mammalian embryo involves transferring the nucleus from a quiescent donor cell into a suitable recipient cell. The donor cell is quiescent, in that it is caused to exit from the growth and division cycle at G1 and to arrest in the G0 state. Nuclear transfer may take place by cell fusion. The reconstituted embryo may then give rise to one or more mammals. The invention is useful in the production of transgenic mammals as well as non-transgenics of high genetic merit.

Abstract: The present invention provides a mammalian artificial chromosome (MAC), comprising a centromere and a unique cloning site, said MAC containing less than 0.1% of the DNA present in a normal haploid genome of the mammalian cell from which the centromere was obtained. The invention further provides a MAC, wherein the unique cloning site is a nucleic acid sequence encoding a selectable marker. The invention also provides methods of preparing a MAC. In addition, the invention provides methods of stably expressing a selectable marker in a cell, comprising introducing a MAC containing the selectable marker into the cell. The invention also provides a cell containing a MAC expressing an exogenous nucleic acid sequence and a transgenic mammal expressing a selectable marker.

Abstract: Transgenic animals expressing Fc receptors and uses for the animals in testing the efficacy of human antibodies and in generating novel antibodies are described.

Abstract: The use of totipotent embryonic stem cells to provide substantially identical cells for embryo cloning techniques is described. The method includes the culture of loose suspensions of inner cell mass cells of bovine animals to retrieve large populations of stem cells. The invention also describes the use of stem cells in various genetic manipulation techniques.

Abstract: The present invention relates to cloning technologies. The invention relates in part to immortalized and totipotent cells useful for cloning animals, the embryos produced from these cells using nuclear transfer techniques, animals that arise from these cells and embryos, and materials, methods, and processes for establishing such cells, embryos, and animals.

Abstract: An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.