Abstract: The invention concerns HER2-transgenic non-human mammals, animal models for screening drug candidates for the treatment of diseases and disorders associated with the overexpression of HER2. In particular, the invention concerns animal models designed to test drug candidates for the treatment of HER2-overexpressing cancers, including breast cancer, that are not responding or poorly responding to current treatments.

Abstract: A chimeric, non-human animal can be produced by a method that entails providing a microcell that contains one or more foreign chromosomes or fragment(s) thereof and then fusing the microcell with a pluripotent cell, thereby introducing the foreign chromosome(s) or fragment(s) into the latter. The pluripotent cell thus obtained can be used to generate a chimeric, non-human animal, the cells, tissues, and/or progeny of which can be the source of a product, such as an antibody, that is associated with one or more genes on the foreign chromosome(s) or fragment(s).

Abstract: This invention relates to transgenic non-human animals comprising a disrupted endothelial nitric oxide synthase gene. These animals exhibit abnormal wound-healing properties and hypertension. This invention also relates to methods of using the transgenic animals to screen for compounds having a potential therapeutic utility for vascular endothelial disorders, such as hypertension, cerebral ischemia or stroke, atherosclerosis and wound-healing activities. Moreover, this invention also relates to methods of treating a patient suffering from hypertension and wound-healing abnormalities with the compounds identified using the transgenic animals, and methods of making the transgenic animals. A method of treating a wound using nitroglycerin is also provided.

Abstract: The present invention relates to a tumor suppressor gene, termed large tumor suppressor (lats), and methods for identifying tumor suppressor genes. The method provides nucleotide sequences of lats genes, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. In a specific embodiment, the lats protein is a human protein. The invention further relates to fragments (and derivatives and analogs thereof) of lats which comprise one or more domains of a lats protein. Antibodies to lats, its derivatives and analogs, are additionally provided. Methods of production of the lats proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Therapeutic and diagnostic methods and pharmaceutical compositions are provided. The invention also relates to recombinant plants and animals and methods of increasing the growth of edible plants and animals.

Abstract: Methods and compositions for introducing a nucleic acid into the genome of at least one cell of a multicellular organism are provided. In the subject methods, a Sleeping Beauty transposon that includes the nucleic acid is administered to the multicellular organism along with a source of a Sleeping Beauty transposase activity. Administration of the transposon and transposase results in integration of the transposon, as well as the nucleic acid present therein, into the genome of at least one cell of the multicellular organism The subject methods find use in a variety of different applications, including the in vivo transfer of genes for use in, among other applications, gene therapy applications.

Abstract: The present invention provides mice that have had their PTP-1B genes disrupted by targeted homologous recombination. The mice have no detectable PTP-1B protein, yet appear to be physiologically normal. However, in the fed state on a normal diet, the mice have half the level of circulating insulin as their wild-type littermates. In glucose and insulin tolerance tests, the mice show an increased insulin sensitivity. When fed a high fat, high carbohydrate diet, the mice show a resistance to weight gain as compared to their wild-type littermates. Methods making the mice and cell lines derived from the mice are also provided. The present invention also provides methods of identifying inhibitors of the enzymatic activity of PTP-1B as well as inhibitors identified by such methods.

Abstract: A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

Abstract: A bigenic mouse is provided whose germ cells and somatic cells contain (i) inactive mouse TAU genes, and/or (ii) a transgene encoding the human TAU gene, with the transgene including all regulatory elements of the human TAU gene necessary for neuronal expression of the transgene in the bigenic mouse, and/or for human patterns of expression of the transgene in the bigenic mouse. The mice of the invention may contain one or two alleles for the human TAU gene (i.e., one or two TAU alleles). Mice of the invention are useful as a source of human Tau protein, and are useful as a model of Alzheimer's, Frontal Temporal Dementia and Parkinson's-like diseases.

Abstract: A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

Abstract: An improved method of gene targeting, referred to as PCR-based gene targeting is disclosed, which generates cell lines or mice in which at least one allele of a specific gene is disrupted by double homologous recombination of a PCR-derived targeting vector with chromosomal DNA. The method is especially applied to murine macrophage cytokine-inducible nitric oxide synthase (MøiNOS).

Abstract: Transgenic organisms (in particular, transgenic animals or plants) bearing positive and/or negative selectable markers are described along with their use in various methods including tissue/cell culture techniques, methods of making monoclonal antibodies, methods of selectively eliminating or depleting a particular tissue/cell type, methods of screening compounds for pharmacological activity and methods of determining the effect of a deficit in a first class of cells or the characteristics of a second class of cells in an organism.

Abstract: The use of a circular vector DNA to produce a pharmaceutical agent for the treatment of mammals or humans by gene therapy wherein the vector DNA contains a selection marker gene and a DNA sequence that is heterologous for the vector which causes a modulation, correction or activation of the expression of an endogenous gene or the expression of a gene introduced into the cells of the mammal or the human by the vector DNA which is characterized in that the vector nucleic acid a) is amplified under selection pressure and cleaved in such a way that the said selection marker gene and the said heterologous DNA are present on separate DNA fragments, b) the DNA fragment which contains the heterologous DNA or both fragments are recircularized to form vectors, c) the DNA fragments are separated before or after the recircularization d) the recircularized DNA fragment which contains the heterologous DNA is isolated and e) the recircularized DNA fragment obtained in this manner is used to produce the pharmaceutical a

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 features a method of promoting an alteration at a selected site in a target DNA, e.g., in the chromosomal DNA of a cell. The method includes providing, at the site: (a) a double stranded DNA sequence which includes a selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or a functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., an agent which inactivates Ku such as an anti-Ku antibody or a Ku-binding oligomer or polymer, and allowing the alteration to occur. The agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent such as an anti-Ku antibody, is preferably provided locally. Components (a), (b), and (c) can be introduced together, which is preferred, or separately.

Abstract: The invention provides transgenic nonhuman mammals producing phosphorylated lysosomal proteins in their milk, and methods of generating the same. Phosphorylation occurs at the 6′ position of a mannose side chain residue. Also provided are methods of purifying lysosomal proteins from milk, and incorporating the proteins into pharmaceutical compositions for use in enzyme replacement therapy.

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: The present invention relates to methods for facilitating site directed homologous recombination in a eukaryotic organism to produce genomic mutants using transposon mediated mutagenesis of cosmid vectors carrying large genomic inserts from the target eukaryotic organism. The transposon carries a bifunctional marker that can be used for selection in both bacteria and the target eukaryotic organism. Minimization of the length of the cosmid vector allows for maximization of the size of the genomic insert carried by the cosmid. Maximization of the size of the genomic insert increases the frequency of homologous recombination with the genome of the target eukaryotic organism.

Abstract: Methods and compositions for transforming cells, resulting in efficient and stable site-specific integration of transgenes, are disclosed. Transformation is achieved by providing an acceptor DNA containing two inverted lox sequences and a donor DNA containing the same two inverted lox sequences, and then contacting the acceptor and donor DNA with a recombinase (e.g., Cre or Flp) which causes recombination at the lox sequences contained in the DNAs. Prior to recombination, the acceptor DNA is preferably integrated into the genome of a cell, such as an embryonic stem cell or a fertilized egg. The acceptor DNA optionally may further contain a negatively selectable marker to allow for screening of cells which have undergone the desired site-specific recombination (e.g., DNA cassette exchange).

Abstract: Methods of unencrypting trait encrypted gene sequences to provide unencrypted RNAs or polypeptides. The invention also relates to methods of encrypting traits including splitting genes between two parental organisms or between a host organism and a vector. The gene sequences are unencrypted when the two parental organisms are mated or when the vector infects the host organism by trans-splicing either the split RNAs or split polypeptides upon expression of the split gene sequences. The invention also includes methods of providing multiple levels of trait encryption and reliable methods of producing hybrid organisms. Additional methods include those related to unencrypting engineered genetic elements to provide polypeptide functions and those directed at recombining non-overlapping gene sequences. The invention also includes integrated systems and various compositions related to the disclosed methods.

Abstract: A process for providing a recombinant, heterologous gene in the genome of a eukaryotic cell is provided. In particular, heterologous DNA is inserted into a recipient gene of the eukaryotic cell by homologous recombination. Moreover, a transgenic or chimeric animal comprising cells with DNA inserted into its genome by homologous recombination is disclosed. Further, the method of making these transgenic animal is taught.

Abstract: The present invention relates to a process for targeted replacement of at least a part of an endogenous gene by at least a part of a foreign gene or targeted insertion of at least a part of a foreign gene at a targeted site in an endogenous gene in a cell by homologous recombination.

Abstract: The present invention relates to an improved and efficient method for simultaneous monitoring of abundance of individual mutants of a microbe in mixed populations where insertion of a known transposon in the genome of a microbe such that each mutant caries a single transposon insertion, isolating the genomic DNA of the mixed population of mutants, fragmenting the same with a frequently cutting restriction enzyme, ligating a double standard adapter to the genomic DNA fragments, amplifying the DNA fragments adjoining transposon insertions thereby generating a set of DNA fragments corresponding only to mutated genes resolving the said amplified DNA fragments followed by comparing the intensity of DNA fragments obtained from population of mutants before selection with that obtained from the population subjected to selection and finally sequencing the DNA fragments that change in abundance to identify the mutated genes.

Abstract: The invention relates to compositions and methods for targeting sequence modifications in one or more genes of a related family of genes using enhanced homologous recombination techniques. These techniques may be used to create animal or plant models of disease as well as to identify new targets for drug or pathogen screening.

Abstract: Animal model useful for testing potential therapeutic agents for the treatment of neurodegenerative disorders, in particular disorders associated with the presence of Lewy pathology.

Abstract: The present invention provides methods which greatly facilitate the rapidity in which cells and transgenic animals with targeted genes may be generated. The invention hastens the investigation of cells and transgenic animals bearing lowered expression of the targeted gene product, a truncated targeted gene product, a fusion protein of the targeted gene and exogenous DNA, or the expression of a different gene from the locus of the targeted gene whose product has reduced expression levels. Also disclosed is a transgenic animal having Cushing's disease. Also disclosed are diagnostic methods for detecting patients with endocrine disorders, and methods for treating or alleviating the symptoms of endocrine disorders.

Abstract: The invention provides in one embodiment a composition which is a linear DNA molecule having a desired replacement sequence, and second and third sequences substantially homologous to non-identical portions of the gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence and the distal ends having a terminating nucleotide analog at each end of the molecule. Another embodiment of the invention provides methods, by blocking the 3′ ends of transforming DNA with 2′3′ dideoxynucleotides, to reduce the frequency of end-mediated DNA insertion. These methods introduce only one copy of the selectable gene at the target locus to achieve a precise gene disruption, reducing or eliminating undesirable and multiple insertions that occur both non-homologously and at the targeted locus.

Abstract: A method of efficiently sequencing multiple exons from complex genomic DNAs is disclosed. The methodology includes the use of bacterial and bacteriophage-derived artificial chromosomes (BBPACS) in novel gene trapping protocols. Targeted gene trapping by homologous recombination, and random gene trapping with the use of a transposon system are exemplified. Included in the invention are methods of preparing a gene map from BBPAC contigs, the resulting gene maps, methods of constructing a cDNA library from BBPAC contigs, and the resulting cDNA libraries.

Abstract: The present invention provides mice which are deficient in the normal expression of one or more members of the RAR or RXR class of receptors, to mice heterozygous for such deficiency, and to cell lines, preferably pluripotent or totipotent cell lines, which are heterozygous or homozygous for such deficiency. The present invention further provides the use of any of the above mice and cell lines in situations where the absence of at least one RAR or RXR receptors, or the normal expression thereof, is desirable.

Abstract: The invention provides a number of strategies for transferring and/or evolving gene(s) associated with cellular DNA uptake so that they confer or enhance DNA-uptake capacity of a recipient cell. Evolution is achieved by recursive cycles of recombination and screening/selection. One such strategy entails evolving genes that confer competence in one species to confer either greater competence in that species, or comparable or greater competence in a second species. Another strategy entails evolving genes for use as components of cloning vector to confer enhanced uptake of the vector. Other strategies entail evolving viral receptors, viruses, and genes that mediate conjugal transfer.

Abstract: Disclosed are purified and isolated DNA sequences encoding eukaryotic proteins possessing biological properties of inosine 5′-monophosphate dehydrogenase (“IMPDH”). Illustratively, mammalian (e.g., human) IMPDH-encoding DNA sequences are useful in transformation or transfection of host cells for the large scale recombinant production of the enzymatically active expression products and/or products (e.g., GMP) resulting from IMPDH catalyzed synthesis in cells. Vectors including IMPDH-encoding DNA sequences are useful in gene amplification procedures. Recombinant proteins and synthetic peptides provided by the invention are useful as immunological reagents and in the preparation of antibodies (including polyclonal and monoclonal antibodies) for quantitative detection of IMPDH.

Abstract: The present invention provides a dual selection cassette (DSC) comprising first and second DNA segments having homology to a eukaryotic viral vector, positive and negative selection genes, each operably linked to their own promoter, and one or more unique restriction enzyme sites (URES) or sitey-directed homologous recombination sites. The present invention also provides a plasmid, pN/P, comprising an independent positive selection marker gene, an origin of replication, and a dual selection cassette. The dual selection cassette and pN/P plasmid can be used to produce eukaryotic gene transfer vectors without requiring temporally-linked double recombination events or the use of specialized bacterial strains that allow the replication of plasmids comprising defective origins of replication. This method usefully increases the ratio of desired to undesired plasmid and vector constructs. Additionally, this invention provides a method for the creation of eukaryotic viral vector libraries.

Abstract: Highly efficient gene integration or gene replacement in the higher eucaryote including animal cells can be performed by using mutant loxP site having the following properties (a)-(c) in the present invention. (a) a nucleotide sequence wherein, in a wild-type loxP site of the following formula (SEQ ID NO: 1) derived from E.

Abstract: The invention relates to calreticulin-deficient cells. The cells can be either homozygous or heterozygous for the calreticulin mutation.

Abstract: Transgenic non-human animals one having a general deletor construct and a second having a general reporter construct are described. The general deletor animals express a heterologous recombinase under the control of an ubiquitously expressed endogenous promoter. Specifically, the Cre recombinase is inserted into the ROSA26 locus of the mouse. The general reporter animals have a gene which is desired to remove flanked by sites recognized by the is heterologous recombinase. This flanked sequence is operatively associated with a marker gene such that when the gene sequence flanked by sites recognized by the heterologous recombinase is excised, the reporter gene is expressed. When the general deletor mouse is crossed with the general reporter mouse the heterologous recombinase is expressed in essentially all cells of the resultant descendants under the control of the ubiquitous promoter. Expression of the recombinase results in the excision of the desired gene in essentially all cells of the descendant animals.

Abstract: Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Abstract: The present invention relates to a method of identifying drugs or agents which have immuno-suppressive effects through or as a result of their effect on calcineurin, including drugs which affect the calcineurin A&agr; (CNA&agr;) subunit or the calcineurin A&bgr; (CNA&bgr;) subunit. In addition, the present invention relates to a method of identifying drugs which reduce (partially or totally) phosphorylation of the microtubule-associated protein tau, in the nervous system of a mammal; a method of identifying drugs which reduce (partially or totally) paired helical filament formation in the nervous system of a mammal; and a method of identifying drugs which reduce (partially or totally) formation of paired helical filaments, amyloid deposits or both. The present invention also relates to transgenic non-human mammals, such as rodents and particularly mice, which lack a functional calcineurin gene and, thus, have disyrupted calcineurin expression.

Abstract: The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.

Abstract: Novel adenovirus vectors and methods for their use are provided to determine the function of the product(s) of one or more sample nucleic acids. The sample nucleic acids are synthetic oligonucleotides, DNA, or cDNA and encode polypeptides, antisense nucleic acids or GSEs. The sample nucleic acids are expressed in a host by recombinant adenovirus vectors to alter at least one phenotype of the host. The altered phenotype(s) is identified as a means to assign a biological function to the product(s) encoded by the sample nucleic acid(s).

Abstract: A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

Abstract: Methods of modulating, tuning and improving hybridization properties and recombination properties of molecules for use in nucleic acid shuffling procedures, relates recombination mixtures and methods of modulating, tuning, improving and evolving splicing of RNAs and proteins are provided.

Abstract: The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.

Abstract: This invention provides methods for modifying a selected gene in cells of a mammalian skin at one or more locations by delivering to the skin cells an effective amount of a composition having a chimeric RNA-DNA oligonucleotide for causing heritable modifications in the selected gene so that the heritable modifications result in phenotypic changes at the locations of the mammalian skin. The invention specifically provides a method for permanent gene correction of a gene mutation by an RNA-DNA oligonucleotide (RDO) in vivo. By this method, a point mutation in the albino BALB/c mouse tyrosinase gene in vivo has been corrected thereby providing for permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in melanocytes of skin at the treated locations. Both topical application and intradermal injection of this oligonucleotide to mice skin resulted in dark pigmentation of several hairs in localized area.

Abstract: The invention provides a number of strategies for transferring and/or evolving gene(s) associated with cellular DNA uptake so that they confer or enhance DNA-uptake capacity of a recipient cell. Evolution is achieved by recursive cycles of recombination and screening/selection. One such strategy entails evolving genes that confer competence in one species to confer either greater competence in that species, or comparable or greater competence in a second species. Another strategy entails evolving genes for use as components of cloning vector to confer enhanced uptake of the vector. Other strategies entail evolving viral receptors, viruses, and genes that mediate conjugal transfer.

Abstract: A peptide and a related complex for transferring an anionic substance of interest into a cell are disclosed wherein said peptide is a cationic peptide capable of binding to an anionic substance, capable to cause membrane disruption and which does not comprise acidic amino acid, preferably glutamic amino acid.

Abstract: Transgenic mice having a phenotype characterized by the substantial depletion of a mature lymphocytic cell type otherwise naturally occurring in the species from which the transgenic mouse is derived. The phenotype is conferred in the transgenic mouse by a transgene contained in at least the precursor stem cell of the lymphocytic cell type which is depleted. The transgene comprises a DNA sequence encoding a lymphatic polypeptide variant which inhibits maturation of the lymphocytic cell type.

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: The present invention provides a transgenic non-human mammal which lacks a functional UCP2 gene. The UCP-2 deficient transgenic knockout mammal described herein provides a source of cells and animals useful to practice methods for the identification and/or evaluation of agents for their ability to affect signaling in cells, such as pancreatic &bgr;-cells, in which ATP serves a regulatory function. Further aspect of the invention provide a method for the identification of agents (e.g., therapeutic agents) which inhibit UCP2 activity; a method for the identification of agents which mimic UCP2 activity and a method of treating diseases or conditions associated with UCP2 function (e.g., negative regulation or uncoupling activity).

Abstract: Methods of modulating, tuning and improving hybridization properties and recombination properties of molecules for use in nucleic acid shuffling procedures, relates recombination mixtures and methods of modulating, tuning, improving and evolving splicing of RNAs and proteins are provided.

Abstract: The invention provides a number of strategies for transferring and/or evolving gene(s) associated with cellular DNA uptake so that they confer or enhance DNA-uptake capacity of a recipient cell. Evolution is achieved by recursive cycles of recombination and screening/selection. One such strategy entails evolving genes that confer competence in one species to confer either greater competence in that species, or comparable or greater competence in a second species. Another strategy entails evolving genes for use as components of cloning vector to confer enhanced uptake of the vector. Other strategies entail evolving viral receptors, viruses, and genes that mediate conjugal transfer.

Abstract: Methods of modulating, tuning and improving hybridization properties and recombination properties of molecules for use in nucleic acid shuffling procedures, relates recombination mixtures and methods of modulating, tuning, improving and evolving splicing of RNAs and proteins are provided.