Abstract: The present invention relates to the production of proteins in host cells, and, more particularly to host cells containing multiple integrated copies of an integrating vector. Suitable integrating vectors for use in the present invention include retrovirus vectors, lentivirus vectors, transposon vectors, and adeno-associated virus vectors. Methods are provided in which the host cells are prepared by using the integrating vectors at a high multiplicity of infection. The host cells are useful for producing pharmaceutical proteins, variants of proteins for use in screening assays, and for direct use in high throughput screening.

Abstract: The present invention relates to a method for producing a recombinant adeno-associated virus (rAAV), in which at different times a helper construct and also a vector construct are introduced into a suitable cell, and preferably the helper construct does not comprise, in particular with the exception of the AAV promoters, any nucleic acid sequences to which at least a Rep protein can essentially specifically bind and preferably the vector construct comprises ITR sequences in flop orientation. The recombinant adeno-associated viruses produced according to the method of the invention are suitable in particular for producing a tumor cell into which additionally nucleic acids coding for GM-CSF and B7.2 has been introduced, which in turn can be used in the form of a medicament for the treatment of cancers.

Abstract: Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. The enhanced rate of mutation can be further augmented using mutagens. Moreover, the hypermutability of mismatch repair deficient cells can be remedied to stabilize cells or mammals with useful mutations.

Abstract: This invention relates to the development of an expression vector containing an antigenic genomic sequence, which is bound to an aggregated protein-polycationic polymer conjugate. More particularly it relates to the use of the expression vector as an oral DNA vaccine or to induce immune response. In specific embodiments, the expression vector is bound to a protein-polycationic polymer suspension.

Abstract: Recombinant lentiviruses and transfer vectors for transgene delivery as well as methods for gene therapy using such vectors are disclosed. The invention provides a third generation lentiviral packaging system and a set of vectors for producing recombinant lentiviruses, as well as novel tissue specific enhancer and promoter elements useful for optimizing liver specific transgene delivery. The transgene is preferably a blood clotting factor such as human factor IX (hFIX) or human factor VIII (hFVIII) and can be used for treatment of hemophilia.

Abstract: The present invention is directed to isolated nucleic acid molecules encoding a voltage-sensitive sodium channel (VSSC) of Musca domestica, the VSSC being capable of conferring insecticide susceptibility or insecticide resistance to Musca domestica, as well as to the isolated voltage-sensitive sodium channels of Musca domestica encoded thereby. Nucleic acid molecules encoding insecticide susceptible VSSCs and nucleic acid molecules encoding insecticide resistant VSSCs are provided. Methods for increasing or decreasing the expression of functional voltage-sensitive sodium channels in host cells are also provided, as well as methods using the sodium channels. Also provided is a method for isolating other voltage-sensitive sodium channels.

Abstract: A method for regulating expression of a tet operator-linked gene in a cell of a subject is disclosed. In one embodiment, the method involves introducing into the cell a nucleic acid molecule encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in the subject. Alternatively, in another embodiment, the method involves obtaining the cell from the subject, introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence, introducing into the cell a second nucleic acid molecule encoding a tTA, to form a modified cell, administering the modified cell to the subject, and modulating the concentration of a tetracycline, or analogue thereof, in the subject. The first and second nucleic acid molecule can be within a single molecule (e.g.

Abstract: Methods and DNA constructs are provided for detection and manipulation of a target eukaryotic gene whose expression is restricted to certain tissues or specialized cell types. The methods include transforming a cell with a first indicator component under the control of a promoter selected for its restricted expression in a particular cell or tissue. The cell is also transformed with a gene trap vector encoding a second indicator component. The cell is allowed to differentiate to produce specialized cell or tissue which is monitored for expression of both the first and second indicator components, thereby detecting a gene into which the trap vector has integrated which is expressed in the same cell or tissue type as the selected promoter.

Abstract: An in vitro method to conduct genomic replication of the viral genomes of viruses that utilize RNA-dependent RNA polymerase for replication (RDRP viruses), such as HCV. The method employs a construct comprising the 3′ and 5′ untranslated regions (UTRs) of the viral genome which are operably linked on the 5′ and 3′ ends of a reporter sequence, in antisense orientation, such that when viral replication is occurring within the cell which produces RDRP, the reporter protein will be made. The method of the invention provides an efficient means for measuring genomic replication in RDRP viruses, and also for the rapid screening of compounds for their ability to inhibit genomic replication of RDRP viruses, including the Hepatitis C virus (HCV).

Abstract: Provided are methods of modulating the persistence of the expression in a cell of a transgene, such as a transgene in a non-Herpes vector or in at least E4&Dgr; adenoviral vector, and related systems. One method comprises contacting the cell with a non-Herpes vector comprising and expressing a gene encoding HSV ICP0, whereupon expression of HSV ICP0 the persistence of expression of the transgene is modulated. Further provided is a system for modulating the persistence of expression of a transgene, which system comprises a non-Herpes vector comprising (i) a gene encoding HSV ICP0 and (ii) a transgene, wherein the HSV ICP0 modulates the persistence of expression of the transgene and either the non-Herpes vector comprises the transgene or the system further comprises a vector, in which case the vector comprises the transgene.

Abstract: Stable cell lines are produced to express high levels of a gene product of interest using VP16, a herpes simplex virus transactivator, and a promoter from herpes simplex virus which is a target for VP16. The transactivator and promoter are introduced to a cell line separately using antibiotic resistance genes as selectable markers on separate vectors.

Abstract: The present invention provides methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell, as well as, enzymes, polypeptides, and a variety of vector constructs useful therefore. In the method, a targeting construct comprises, for example, (i) a first recombination site and a polynucleotide sequence of interest, and (ii) a site-specific recombinase, which are introduced into the cell. The genome of the cell comprises a second recombination site. Recombination between the first and second recombination sites is facilitated by the site-specific recombinase. The invention describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy techniques.

Abstract: An adenovirus vector/packaging cell line system is disclosed, in which the vector replication is blocked by deletion of a single gene, which deletion does not interfere with any other viral functions. The deleted gene is the gene of the adenovirus protease. The protease is expressed in a complementing (packaging) cell line through a regulatable expression cassette which induces no toxic effects in the cells, thus making the generation and propagation of the vector easier and more efficient. As the deleted gene is highly specific of adenovirus, no complementation of the gene in transduced cells is expected, which increases the safety of the new vectors for gene transfer purposes. Also disclosed is a new system of generating recombinant adenovirus vectors by positive selection of recombinants deleted for the endogenous protease gene, which gene is cloned in another region of the adenoviral genome.

Abstract: The invention provided improved methods of making and producing recombinant proteins in in vitro cultures of host cells using apoptosis inhibitors. The use of one or more apoptosis inhibitors in the methods can reduce apoptosis in the cell cultures and markedly improve yield of the desired recombinant proteins.

Abstract: The present invention is directed to nucleic acid and amino acid sequences for transformation constructs containing piggyBac or tagalong transposable elements. These constructs allow for the precise excision and insertion of heterologous DNA into a host cell.

Abstract: The present invention provides AAV “split-packaging” genes, and packaging cells comprising such genes, for use in the production of high titers of replication-incompetent recombinant AAV vectors that can be used to deliver transgenes of interest to a variety of mammalian cells.

Abstract: The present invention relates to novel fusion proteins, DNA molecules encoding the same, vectors comprising the DNA molecules, and host cells containing the vectors for use in measuring protease activity using a novel transcriptional assay. This invention also relates to a method for determining the inhibitory activity of a compound against a protease and to a method for comparing the activity of two proteases which recognize the same cleavage site. Kits for assaying protease activity comprising DNA molecules encoding the fusion protein substrates of this invention are also contemplated.

Abstract: A recombinant lentiviral vector expression system comprises a first vector that comprises a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome. The vector contains at least one defect in at least one gene encoding an EIAV structural protein, but is preferably a gag/pol expression vector. The expression system further comprises a second vector, also comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, and additionally containing a multiple cloning site wherein a heterologous gene may be inserted. The expression system also comprises a third vector which expresses a viral envelope protein. The first and third vectors are packaging signal-defective. When the expression system is transfected into a lentivirus-permissive cell, replication-defective EIAV particles may be produced, which particles are useful in delivering heterologous genes to a broad range of both dividing and non-dividing cells.

Abstract: The present invention relates to methods and compositions for the production of recombinant Adeno-Associated Viruses (rAAV). In particular, the invention discloses nucleic acid constructs and packaging cells having improved properties for rAAV production, as well as novel methods of titration and characterization of rAAV preparations. The invention also describes novel sequences which promote or increase the packaging of nucleic acids in rAAV, and their use for producing rAAV with high efficiency. The invention can be used for producing or testing high quality rAAV preparations, for biological, preclinical, clinical or pharmaceutical uses.

Abstract: Vectors of the invention facilitate selection of host cells having operably incorporated query genes, and substitution of the query gene with a different gene.

Abstract: The invention provides a system for production of retroviruses which are replication incompetent. In the system, gag and pol retroviral structural proteins are expressed separately by different plasmids in a packaging cell line. Separate gag and pol expressing plasmids are provided, as are packaging cell lines containing such plasmids. Retrovirus products of the retroviral vector production system, including chimeric retroviruses, are also provided.

Abstract: A nucleic acid having a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme, and an inducible promoter operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3′ sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible, is described. A cell containing the nucleic acid and methods of using the cell are also described.

Abstract: The invention relates to a process for recombinantly preparing picornavirus particles, in particular hepatitis A virus particles, their precursors and particles which are derived therefrom, with a structural protein precursor molecule (P1-2A or P1) and the corresponding P3 region (3ABCD) being coexpressed in cis or in trans.

Abstract: Novel cell lines useful for trans-complementing E1-deleted adenoviral vectors are described. The cell lines are capable of providing high yields of E1-deleted adenoviral vectors in the absence of replication-competent adenovirus over multiple passages.

Abstract: The present invention provides compositions and methods of producing recombinant AAV (rAAV) virions in large amounts or high titers. Also provided are methods for producing stably transformed host cells capable of producing rAAV virions.

Abstract: A gene is transferred to the early stage chicken embryo by electroporation with placing the electrode tips to hold both ends of the embryo. Transfer of the desired gene to the early stage embryo was detected using the &bgr;-galactosidase gene of E coli as a reporter gene. The expression of the gene in the early stage embryo of chicken was thus confirmed. This method for transferring a gene into undifferentiated cells is simple to implement, highly efficient in transferring a gene, and applicable to various animal species.

Abstract: The present invention provides an immortalized insulin producing human &bgr;-cell which may be rendered glucose responsive by suitable bioengineering methods. The invention also provides a method for producing an immortalized glucose responsive insulin producing human &bgr;-cell comprising the steps of selecting an unregulated immortalized human insulin secreting &bgr;-cell, transecting said selected cell line with elements for the genetic control of glucose responsiveness and proliferating said transfected &bgr;-cell accordingly.

Abstract: This invention provides a mammalian cell useful for retroviral packaging comprising two plasmids, both of which comprise the 5′ long terminal repeat (LTR) sequence from a helper virus, neither of which comprise a functional &psgr; packaging sequence or a 3′ LTR from the helper virus, one of which comprises the env gene from the helper virus and the other of which comprises the gag and pol genes from the helper virus. This invention also provides a process for preparing a producer cell useful for transferring a foreign gene into a mammalian cell which comprises treating the above-described mammalian cell with a vector plasmid so as to insert the vector plasmid into the cell and thus create the producer cell, the vector plasmid comprising the foreign gene, a functional &psgr; packaging sequence from the helper virus, both the 5′ and 3′ LTRS from the helper virus, and a gene encoding a selectable or identifiable phenotypic trait, and recovering the producer cell so created.

Abstract: A method is provided for inducing DNA synthesis in differentiated neurons. According to certain embodiments of the invention, a method for inducing DNA synthesis in a differentiated neuron is provided that includes obtaining a vector comprising nucleic acid encoding an E2F regulator and/or an E1A regulator, wherein the vector can be used to express the nucleic acid in a differentiated neuron, and transfecting a differentiated neuron with the vector.

Abstract: Novel cell lines useful for trans-complementing E1-deleted adenoviral vectors are described. The cell lines are capable of providing high yields of E1-deleted adenoviral vectors in the absence of replication-competent adenovirus over multiple passages.

Abstract: Novel packaging cell lines which produce recombinant retrovirus, free of detectable helper-virus are disclosed. Also disclosed are methods of making the cell lines and methods of producing recombinant retroviruses from the cell lines. Retroviruses produced by the cell lines include lentiviruses, such as HIV, capable of transfering heterologous DNA to a wide range of non-dividing cells. The packaging cells contain at least three vectors which collectively encode retroviral gag, pol, and env proteins, wherein the gag and pol genes are separated, in part, onto two or more different vectors. This is made possible by fusing Vpr or Vpx to pol proteins separated from gag so that the proteins are targeted to assembling virions. Among other advantages, the packaging cells provide the benefit of increased safety when used in human gene therapy by virtually eliminating the possibility of molecular recombination leading to production of replication-competant helper virus.

Abstract: Materials and methods of activating T lymphocytes with specificity for particular antigenic peptides are described, as well as the use of activated T lymphocytes in vitro for the treatment of a variety of disease conditions. In particular, a method for producing a synthetic antigen presenting drosophila cell line for activating T lymphocytes to a specific peptide is described.

Abstract: This invention provides helper-dependent adenovirus cloning vectors and helper adenoviruses, and methods for making and using such preparations, wherein the helper adenoviruses contain recombinase target sites that are useful in reducing the level of contamination of helper virus in helper-dependent adenovirus vector preparations.

Abstract: The invention is directed to novel systems for the high level production of purified recombinant adeno-associated virus (rAAV) vector stocks comprising producer cell lines and helper adenoviruses. These systems provide high level production of rAAV vector stocks that are not contaminated by helper viruses or have very minimal contamination with helper virus. The invention is also directed to methods for the production of high yield, purified rAAV vector stocks using the systems of the invention.

Abstract: The invention relates to a method for the preparation of DNA virus vectors capable of replication in eukaryotic as well as in prokaryotic cells as well as to DNA virus vectors prepared by this method. Preferably, the method is used to prepare Epstein-Barr virus vectors.

Abstract: The present invention relates to methods and compositions for tissue-restricted gene recombination. In particular, the present invention provides methods and compositions for tissue-restricted gene recombination in post-mitotic cells. The present invention further provides methods for gene recombination in post-mitotic cells comprising the delivery of a Cre recombinase to the target tissue to facilitate recombination in a desired target nucleic acid.

Abstract: The invention relates to methods for targeting an exogenous polynucleotide or exogenous complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a eukaryotic cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active eukaryotic cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active eukaryotic cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell.

Abstract: A method of reducing immune response to a viral vector containing a selected transgene is provided. The method involves co-administration of the viral vector and a selected immune modulator capable of inhibiting the formation of neutralizing antibodies and/or CTL elimination of the vectors upon repeated administration.

Abstract: Disclosed are isolated transposable elements, or isolated DNA sequences which encode a transposase protein (or a portion of a transposase protein). The isolated transposable elements or the isolated DNA sequences being characterized by the ability to hybridize to the DNA sequence of Minos-1. The invention also relates to a purified transposase protein, or peptide fragments thereof, encoded by such DNA sequences. Such transposable are useful in methods for the stable introduction of a DNA sequence of interest into a cell. The invention further relates to transgenic animals, gene tagging and insertional mutagenesis produced by such methods. The sequence information disclosed herein is useful in the design of oligonucleotide primers which are useful for the isolation of related members of the Tc-1 family of transposable elements.

Abstract: The present invention is directed to nucleic acid and amino acid sequences for transformation constructs containing piggyBac or tagalong transposable elements. These constructs allow for the precise excision and insertion of heterologous DNA into a host cell.

Abstract: This invention provides monoclonal antibodies that bind to both E-selectin and to P-selectin, and inhibit the binding of these proteins to counterreceptors. The invention also provides nucleic acids encoding these antibodies and methods for using the antibodies in the treatment of inflammatory conditions.

Abstract: Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Abstract: This invention provides a method for preparing a hybridoma cell line which produces a monoclonal antibody which specifically recognizes and binds to a tumor associated antigen which comprises: (a) cotransfecting a CREF-Trans 6 cell line with DNA isolated from a neoplastic, human cell and a plasmid which encodes a selectable or identifiable trait; (b) selecting transfected cells which express the selectable or identifiable trait; (c) recovering the cells so selected in step (b); (d) injecting the cells so recovered in step (c) into a suitable marine host; (e) maintaining the resulting first murine host for a period of time effective to induce the cells injected in step (d) to form a tumor in the murine host; (f) isolating the tumor formed in step (e); (g) obtaining tumor cells from the isolated tumor in step (f); (h) coating the tumor cells obtained in step (9) with an antiserum generated against the CREF Trans-6 cell line (i) injecting the antiserum-coated cells from step (h) into a plurality of suitable seco

Abstract: A simple method for modifying genes in a recombination deficient host cell is disclosed. Such modifications include generating insertion, deletions, substitutions, and/or point mutations at any chosen site in the independent origin based cloning vector. The modified gene can be contained in an independent origin based cloning vector that is used to introduce a modified heterologous gene into a cell. Such a modified vector may be used in the production of a germline transmitted transgenic animal, or in gene targeting protocols in eukaryotic cells.

Abstract: The present invention relates to novel fusion proteins, DNA molecules encoding the same, vectors comprising the DNA molecules, and host cells containing the vectors for use in measuring protease activity using a novel transcriptional assay. This invention also relates to a method for determining the inhibitory activity of a compound against a protease and to a method for comparing the activity of two proteases which recognize the same cleavage site. Kits for assaying protease activity comprising DNA molecules encoding the fusion protein substrates of this invention are also contemplated.

Abstract: The present invention is directed to compositions and methods for a genetic system of detecting protein--protein interactions in a mammalian host cell. Two fusion proteins are made in the host cell. The first fusion protein contains a DNA binding domain which is fused to a so-called bait protein. The second fusion protein consists of a transcriptional activation domain fused to a so-called test protein. The transcriptional activation domain is recruited to the promoter through the functional interaction between the bait protein and the test protein. Subsequently the transcriptional activation domain interacts with the basal transcription machinery to activate expression of one or more reporter genes which can be identified and characterized. The individual compositions are useful for analyzing protein--protein interactions between known proteins and to isolate, clone and characterize unknown proteins. The individual compositions can be used to express the fusion proteins either transiently or stably.

Abstract: Genetically engineered cells are provided which can serve as universal donor cells in such applications as reconstruction of vascular linings or the administration of therapeutic agents. The cells include a coding region which provides protection against complement-based lysis, i.e., hyperacute rejection. In addition, the cell's natural genome is changed so that functional proteins encoded by either the class II or both the class I and the class II major histocompatibility complex genes do not appear on the cell's surface. In this way, attack by T-cells is avoided. Optionally, the cells can include a self-destruction mechanism so that they can be removed from the host when no longer needed.

Abstract: A method is provided for inducing DNA synthesis in differentiated neurons. According to certain embodiments of the invention, a method for inducing DNA synthesis in a differentiated neuron is provided that includes obtaining a vector comprising nucleic acid encoding an E2F regulator and/or an E1A regulator, wherein the vector can be used to express the nucleic acid in a differentiated neuron, and transfecting a differentiated neuron with the vector.

Abstract: Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of this invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the .zeta.

Abstract: Described are preferred myocardial grafts of skeletal myoblasts or cardiomyocytes, and cellular compositions and methods useful in obtaining the grafts. The myocardial grafts are stable and can be used, for example, to deliver recombinant proteins directly to the heart.