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: Methods to find optimal integration sites within a plant genome are provided. More particularly, a plant is transformed with a target site having an expression cassette comprising a nucleotide sequence operably linked to a promoter active in the plant. The target site is flanked by non-identical recombination sites, Transformed protoplast, tissues, or whole plants can be tested to determine the levels of activity of the inserted gene, By comparison of cellular activities of the gene in different insertion sites, preferred integration sites may be found wherein the gene is expressed at high or acceptable levels. These plants can then be utilized with subsequent retargeting techniques to replace the nucleotide sequence with other genes or nucleotide sequences of interest contained in a transfer cassette.

Abstract: Compositions, kits, and methods are provided for use in a recombinational cloning or subcloning methods for constructing expression vectors which comprise: ligating a library of double-stranded linear donor DNAs, where each member of the library includes a donor DNA sequence, with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the single circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector, the circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.

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: Compositions and methods for introducing a DNA of interest into a genomic target site are provided. In particular, the methods and compositions involve the use of a combination of target sites for two site specific recombinases and expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities to multiple target sites, and nucleotide sequences and expression cassettes encoding these recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with a DNA of interest that is flanked by corresponding target sites. Expression of the recombinase results in integration of the DNA of interest into the genome of the cell. The compositions and methods of the invention have use in the construction of stably transformed eukaryotic cells, and in particular, plant cells.

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: 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: Methods, transformation constructs, and transgenic animals for identifying anti-tumor agents and anti-tumor drug targets are described. The transformation constructs are used to generate transgenic animals that have altered expression of an oncogene or tumor suppressor gene in a target tissue that is dispensable for viability and reproduction. In some embodiments, the altered expression results in abnormal proliferation of the target tissue and normal proliferation in all other tissues. Anti-tumor drug targets can be identified by generating progeny of the transgenic animals that have mutations in various genes. Gene mutations that result in a specific reduction or killing of the target tissue are identified as possible anti-tumor drug targets and are further evaluated. Anti-tumor agents are identified that mimic the effect of the gene mutations that result in specific reduction of the target tissue.

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: 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: 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: A method of expression DNA in a cell comprises, in a cell that expresses a replication VpA factor, transfecting the cell with a vector, wherein (i) the vector contains a DNA, or is adapted to receive a DNA, in operative combination with a promoter for expression of the DNA; and (ii) extrachromosomal replication of the vector is dependent upon presence within the cell of the replication factor. Stable El maintenance of the vector and expression of the DNA is thereby achieved. Vectors for expression of DNA are provided as are assays for the effect of expression of DNAs in cells, the DNAs being H expressed from the vectors.

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: The present disclosure describes a biologically active modified urokinase and high resolution crystalline forms of modified urokinase. Polynucleotides which encode modified urokinase and methods for making modified urokinase are also disclosed.

Abstract: Methods for efficient production of recombinant AAV are described. In one aspect, three vectors are introduced into a host cell. A first vector directs expression of cre recombinase, a second vector contains a promoter, a spacer sequence flanked by loxP sites and rep/cap, and a third vector contains a minigene containing a transgene and regulatory sequences flanked by AAV ITRs. In another aspect, the host cell stably or unducibly expresses cre recombinase and two vectors carrying the other elements of the system are introduced into the host cell.

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: The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in ES cells transmitted into the mouse germ line. These chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Additionally, translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in organisms such as Drosophila. Mice with defined regions of segmental haploidy are useful for genetic screening and allow accurate models of human chromosomal diseases to be generated.

Abstract: The invention concerns a method for producing recombinant virus. This method is based on the use of baculovirus for providing the complementary functions. It also concerns constructs used for implementing this method, the producing cells, and the resulting virus.

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: Novel helper vectors are provided for complementing defective recombinant viral vectors, characterized in that they are provided with recombination sequences recognized by a recombinase. A complementation cell expressing the recombinase, and a method for preparing recombinant viral vectors as infectious viral particles for transferring and expressing genes of interest in a host organism or cell, are also provided. The invention is particularly suitable for use in gene therapy, especially in humans.

Abstract: A transposition assembly for the transfer of a DNA fragment of interest into the ribosomal nuclear DNA of an eukaryotic cell. An insertion means, an eukaryotic cell and a pharmaceutical composition comprising said transposition assembly, as well as a method for the in vitro transfer of said DNA fragment, are also disclosed.

Abstract: A method of analysis of concerted integration in which viral integrase enzyme is first incubated with donor DNA molecules followed by incubation with target DNA molecules. The donor DNA molecule having at least one unique restriction site for analysis of concerted integration product.

Abstract: A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a transposase protein and (b) a polynucleotide that comprises (1) a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and (2) a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Abstract: In accordance with the present invention, there are provided targeted loss of function mutant mice which express less than endogenous levels of at least one member of the steroid/thyroid superfamily of receptors in at least one specific tissue type. For example, mutations in the RXR&agr; gene in mouse germlines are lethal in the embryonic stage between E13.5 and E16.5 when bred to homozygosity. The major defect responsible for this lethal effect is hypoplastic development of the ventricular chambers of the heart, which is manifest as a grossly thinned ventricular wall with concurrent defects in ventricular septation. This phenotype is identical to a subset of the effects of embryonic vitamin A deficiency, and therefore establishes RXR&agr; as a genetic component of the vitamin A signaling pathway in cardiac morphogenesis.

Abstract: Methods for efficient production of recombinant AAV are described. In one aspect, three vectors are introduced into a host cell. A first vector directs expression of cre recombinase, a second vector contains a promoter, a spacer sequence flanked by loxP sites and rep/cap, and a third vector contains a minigene containing a transgene and regulatory sequences flanked by AAV ITRs. In another aspect, the host cell stably or inducibly expresses cre recombinase and two vectors carrying the other elements of the system are introduced into the host cell.

Abstract: An adenovirus E1/E4 expressing packaging cell line is provided, which permits the generation of recombinant adenoviruses deleted in both gene regions. A method for enhancing the efficiency of transduction of a recombinant AAV into a target cell is provided by infecting a target cell with a recombinant AAV comprising a selected transgene under the control of regulatory sequences. The infected cell is contacted with an agent which facilitates the conversion of single stranded recombinant virus to its double stranded form.

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: 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 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: 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: The subject invention finds utility in the area of gene therapy of diseases. More specifically, the invention concerns the making of a novel non-viral vector which can bind to desired DNA to form a combination useful to transfect diseased mitochondria of human or animal cells. The non-viral vector comprises a dequalinium salt subjected to standard liposome production procedures to obtain the vector named DQAsomes.

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: The invention provides a novel retroviral packaging system, in which retroviral packaging plasmids and packagable vector transcripts are produced from high expression plasmids after stable or transient transfection in mammalian cells. High titers of recombinant retrovirus are produced in these transfected mammalian cells and can then transduce a mammalian target cell by cocultivation or supernatant infection. The methods of the invention include the use of the novel retroviral packaging plasmids and vectors to transduce primary human cells, including T cells and human hematopoietic stem cells, with foreign genes by cocultivation or supernatant infection at high efficiencies. The invention is useful for the rapid production of high titer viral supernatants, and to transduce with high efficiency cells that are refractory to transduction by conventional means.

Abstract: The invention is based on the discovery that recombinagenic oligonucleobases are active in prokaryotic cells that contain a strand transfer activity (RecA) and mismatch repair activity (MutS). Using this system a type of Duplex Mutational Vector termed a Heteroduplex Mutational Vector, was shown to be more active in prokaryotic cells than the types of mutational vectors heretofore tested. Further improvements in activity were obtained by replacing the tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2′-O-methyl-uridine, to link the two strands of the recombinagenic oligonucleobase and removing the DNA-containing intervening segment. The claims concern Duplex Mutational Vectors that contain the above improvements. In an alternative embodiment the claims concern the use of Duplex Mutational Vectors in prokaryotic cells.

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: A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

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: A method for generating adenoviral vectors from polynucleotides such as plasmids wherein there occurs recombinase-mediated transfer of an adenoviral ITR and terminal proteins bound to the ITR to a plasmid, thus enabling the plasmid to replicate as an adenoviral vector. Such method enables the rapid generation of adenoviral vectors at high titers from plasmids without the use of selectable markers and screening procedures. Such method enables the rapid generation of adenoviral vectors devoid of adenovirus backbone genes. The method also may be employed to generate hybrid adenoviral-retroviral vectors that convert transduced cells into producer cells that produce retroviral vectors to effect high level, permanent genetic modification of cells in vivo.

Abstract: The present invention provides isolated adenovirus-associated viruses ("AAV"), including AAV isolates designated AAV3B and AAV6. The invention also provides nucleic acid molecules of AAV3B (SEQ ID NO: 1) or AAV6 (SEQ ID NO: 2), including DNA or RNA, and provides substantially purified polypeptides encoded by AAV3B or AAV6, as well as antibodies specific for such polypeptides. The invention also provides infectious AAV3B and AAV6 clones; AAV viral vectors, which can be hybrid AAV viral vectors; AAV vector plasmids; and AAV helper plasmids; each comprising at least a portion of an AAV3B (SEQ ID NO: 1) or AAV6 (SEQ ID NO: 2) nucleic acid molecule. The invention further provides host cells containing at least a portion of an AAV3B or AAV6 nucleic acid molecule and progeny cells derived therefrom, and provides non-human transgenic mammals having an AAV vector genome stably integrated in a chromosome.

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 features mouse models for Nkx-2.2 gene function and for Nkx-6.1 gene function, wherein the transgenic mouse is characterized by having a defect in Nkx-2.2 gene function or a defect in Nkx-6.1 gene function (where, because Nkx-2.2 acts upstream of Nkx-6.1, a defect in Nkx-2.2 gene function affects Nkx-6.1 gene function) and by having a decreased number of insulin-producing cells relative to a normal mouse. Where the transgenic mouse contains a defect in Nkx-2.2 gene function, the mouse is further characterized by a decreased number of serotonin-producing cells relative to a normal mouse. The transgenic mice may be either homozygous or heterozygous for the Nkx-2.2 or Nkx-6.1 defect.

Abstract: An assay is provided for determining the presence of contaminating microbial host cells in a culturing vessel containing a microbial host cell strain that utilizes more than one carbohydrate source as a substrate comprising:(a) culturing the microbial host cell strain in the vessel, wherein the strain comprises nucleic acid encoding a polypeptide of interest and is genetically marked so as not to utilize a carbohydrate source as a substrate;(b) plating an isolated sample of culture from step (a) onto culture media supplemented with a carbohydrate source not utilized by the host cell strain as a substrate;(c) incubating the plated cells at a temperature and for a time sufficient for any positive colony to grow to a detectable level; and(d) detecting whether any colonies are growing on the supplemented culture media.

Abstract: The present invention is directed to mice which are genetically altered to be deficient in the normal expression of RXR.gamma., 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 invention further provides mice and cell lines which, in addition to being deficient in RXR.gamma., are genetically altered to be deficient in the expression of RXR.alpha. and/or RXR.beta.. The present invention further provides the use of any of the above mice and cell lines in situations where the absence of RXR.gamma., or the normal expression thereof, is desirable.

Abstract: The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in ES cells transmitted into the mouse germ line. These chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Additionally, translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in organisms such as Drosophila. Mice with defined regions of segmental haploidy are useful for genetic screening and allow accurate models of human chromosomal diseases to be generated.

Abstract: The present invention provides novel elements for improving genetic engineering techniques for producing recombinant nucleic acid molecules and/or recombinant cells. The novel elements are capable of integrating desired nucleic acid material into other nucleic acid materials, specifically into the genome of a host cell. The novel elements are derived from or based on transposons, in particular from the Tc/Mariner superfamily. In particular, the essential elements of Tc1 enabling excision and pasting of the desired nucleic acid material are provided, together with the relevant transposase activity in cis or in trans.

Abstract: Selectable Herpesvirus saimiri vectors which have a selection gene inserted into a junction region of the L- and H-DNA are described. Vectors of this type are able persistently to infect human T cells and thus are suitable for the expression of foreign genes in human T cells. An additional advantage is that no infectious virus particles are produced during this.

Abstract: The present disclosure provides retrotransposons and retrotransposon derivatives and methods for their uses. Specifically, the present invention provides Ty5-6p and derivatives. Ty5-6p and its derivatives integrate preferentially in the genome of eukaryotes in silent chromatin and in regions like silent chromatin. Ty5-6p insertions can be used to regulate the life span of cells, to genetically mark cells, to deliver gene therapy and to identify genes involved in development and in senescence.