WO2002003790A2 - Souris transgeniques contenant des interruptions de genes kinase cibles - Google Patents

Souris transgeniques contenant des interruptions de genes kinase cibles Download PDF

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WO2002003790A2
WO2002003790A2 PCT/US2001/021821 US0121821W WO0203790A2 WO 2002003790 A2 WO2002003790 A2 WO 2002003790A2 US 0121821 W US0121821 W US 0121821W WO 0203790 A2 WO0203790 A2 WO 0203790A2
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gene
target gene
cell
agent
cells
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WO2002003790A3 (fr
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Keith D. Allen
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Deltagen, Inc.
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Publication of WO2002003790A3 publication Critical patent/WO2002003790A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.
  • Protein kinases are a large family of proteins that share a homologous catalytic domain of 250 to 300 amino acids, which can be subdivided into 11 subdomains. Protein kinases frequently play key roles in the normal regulation of growth and development in eukaryotic organisms. As a consequence, aberrant expression or mutations in this family of molecules frequently result in transformation.
  • NEKs a newly-emerging subfamily of kinases of which the founding member is the NIMA kinase of Aspergillus nidulans.
  • the Aspergillus nidulans 'never in mitosis A' (NIMA) gene encodes a serine/threonine kinase that controls initiation of mitosis.
  • NIMA-related kinases NEKs are a group of protein kinases that are homologous to NIMA.
  • NIMA is essential for passage of Aspergillus cells from G2 into mitosis, although its substrates and mode of action remain to be determined. In Aspergillus, NIMA cooperates with the CDK1 protein kinase to promote entry into mitosis, and overexpression of NIMA in a variety of heterologous species promotes a premature onset of chromosome condensation and, in some cases, mitotic spindle formation. This has been interpreted to suggest evolutionary conservation of a pathway involving
  • NDVIA-related kinases kinases structurally-related to NDVIA have been identified in many species, although the functional relationship between vertebrate NEKs and fungal NIMA remains uncertain.
  • Northern blot analysis detected 1.6-, 2.6-, and 9.5-kb NEK6 transcripts. The 1.6-kb transcript was expressed at highest levels in liver and placenta.
  • Li et al. (1999) mapped the NEK6 gene to 9q33.3-q34.11.
  • NEK6 and NEK7 Two novel murine protein kinase genes, designated NEK6 and NEK7, which are highly similar to each other (87% amino acid identity in the predicted kinase domain).
  • NEK6 and NEK7 are also highly similar to the F19H6.1 protein kinase of Caenorhabditis elegans (76 and 73% amino acid identity in the kinase domain, respectively), and phylogenetic analysis suggests that these three proteins constitute a novel subfamily within the NDVIA family of serine/threonine kinases. In contrast to the other documented NIMA-related kinases, NEK6/7 and F19H6.1 harbor their catalytic domain in the C-terminus of the protein. Immunofluorescence suggests that NEK6 and NEKk7 are cytoplasmic.
  • Linkage analysis using the murine BXD recombinant inbred strain panel, localized the NEK6 gene to chromosome 2 at 28 cM.
  • Kandli et al. assigned the NEK7 gene to chromosome 1 at approximately 73 cM.
  • the full-length 2249 bp cDNA for the murine NEK6 gene is identified in GenBank as GI/NID number: 10312093 and Accession number: AF218847.
  • PKL12 enzyme possesses intrinsic kinase activity, capable of phosphorylating enolase and promoting autophosphorylation, with a ser/thr specificity.
  • Ligos et al. also constructed the human homolog, STK16, using several human ESTs with sequence similarity to the PKL12 gene. They also identified the putative homologue proteins from Saccharomyces cerevisiae and Arabidoposis thaliana.
  • the STK16 gene encodes a 310-amino acid protein that shares 94% identity with the mouse homologue. Because its size is very close to the theoretical minimal size for a core catalytic domain, the authors suggested that STK16 may constitute the catalytic subunit of a more complex holoenzyme or multisubunit kinase.
  • Northern blot analysis Ligos et al. showed that both STK16 and PKL12 are ubiquitously expressed at very low levels. Expression of an STK16 fusion protein showed that STK16 is localized with Golgi and Golgi-derived vesicles.
  • Krct is expressed preferentially within specific cellular compartments in multiple tissues, in particular within the testis and gastrointestinal tract.
  • Krct is most closely related to four previously undescribed kinases in Saccharomyces cerevisiae, Arabidopsis thaliana and Caenorhabditis elegans.
  • Krct possesses an unusually long 5 '-untranslated region containing multiple upstream initiation codons and, in this regard, is similar to many proto- oncogenes that regulate normal growth and differentiation.
  • Krct is located on mouse chromosome 11 closely linked to the epidermal growth factor receptor and, therefore, is likely to be co-amplified in a variety of human tumors.
  • the full-length 1512 bp cDNA for the murine PKL12 gene bears GenBank GI or NID number: 4063861 and Accession number: AF089869.
  • the present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.
  • the present invention provides transgenic cells comprising a disruption in a targeted gene.
  • the transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination.
  • the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells.
  • the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of the targeted gene.
  • the transgenic cells are derived from the transgenic animals described below.
  • the cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
  • the present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant.
  • the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the targeted gene.
  • the targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct.
  • the targeting construct may also comprise other regulatory elements that may enhance homologous recombination.
  • the present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a target gene.
  • the transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the target gene.
  • the transgenic animals of the present invention are defective in the function of the target gene.
  • the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a target gene.
  • the present invention also provides methods of identifying agents capable of affecting a phenotype associated with a disruption of a target gene in a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a "normal" or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration).
  • the invention further provides agents identified according to such methods.
  • the present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the target gene.
  • the present invention further provides a method of identifying agents having an effect on target gene expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse.
  • the method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control.
  • a control animal which may be, for example, a wild-type animal or alternatively, a transgenic animal control.
  • Compounds that may have an effect on target gene expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
  • the invention also provides cell lines comprising nucleic acid sequences of a target gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the target gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the target gene, comprising the steps of contacting the target gene or target protein with an agent and detecting an agent/ target gene or agent/target protein complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
  • the invention further provides methods of treating diseases or conditions associated with a disruption in a target gene, and more particularly, to a disruption in the expression or function of the target gene or the target protein encoded by the target gene.
  • methods of the present invention involve treating diseases or conditions associated with a disruption in the target gene's expression or function, including administering to a subject in need, a therapeutic agent which effects target gene or target protein expression or function.
  • the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi- synthetic, or recombinant target gene, target gene products or fragments thereof as well as natural, synthetic, semi-synthetic or recombinant analogs.
  • the present invention further provides methods of treating diseases or conditions associated with disrupted targeted gene expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated target genes.
  • gene refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein.
  • the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleo- tides, ribonucleotides and/or their analogs.
  • Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes single-, double-stranded and triple helical molecules.
  • Oligonucleotide refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer” refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme- mediated nucleic acid synthesis.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.
  • Analogs of purities and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylamino- methyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyl- adenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl- adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine.
  • uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
  • a “fragment" of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
  • the term “gene targeting” refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.
  • homologous recombination refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.
  • homologous denotes a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be deteraiined using a "BLASTN” algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
  • target gene refers to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination.
  • the target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes.
  • the target gene comprises a portion of a particular gene or genetic locus in the individual's genomic DNA.
  • the target gene of the present invention in alternate embodiments, is a NEK6 gene or a PKL12 gene.
  • a “NEK6 gene” refers to a nucleotide sequence that encodes a NEK6 protein, such as a sequence comprising SEQ ID NO:l, identified in GenBank as GI NID number: 10312093 and Accession number: AF218847, or a homolog thereof.
  • PLL12 gene refers to a nucleotide sequence that encodes a PKL12 protein, such as a sequence comprising SEQ ID NO: 4, identified in GenBank as GI or NID number: 4063861 and Accession number: AF089869, or a homolog thereof (such as the human homolog of PKL12, STK16).
  • "Disruption" of a target gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes which may be of animal, plant, prokaryotic, or viral origin.
  • transgenic cell refers to a cell containing within its genome a target gene that has been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting.
  • transgenic animal refers to an animal that contains within its genome a specific gene that has been disrupted by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).
  • selectable marker or “positive selection marker” refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo r ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected with the constructs of the present invention.
  • modulates refers to the inhibition, reduction, increase or enhancement of a target gene or target protein function, expression, activity, or alternatively a phenotype associated with a disruption in a target gene. In a preferred embodiment, the term “modulates” refers to agents that act as agonists or antagonists of the target kinase.
  • ameliorates refers to a decreasing, reducing or eliminating of a condition, disease, disorder, or phenotype, including an abnormality or symptom associated with a disruption in a target gene.
  • abnormality refers to any disease, disorder, condition, or phenotype in which a disruption of a target gene is implicated, including pathological conditions.
  • Figure 1 shows a polynucleotide sequence for a murine NEK6 gene (SEQ ID NO: 1).
  • Figures 2 and 3 show the design of the targeting construct used to disrupt an NEK6 gene, as well as the location and extent of the deletion in the target sequence.
  • Figure 3 shows the sequences identified as SEQ ID NO:2 and SEQ ID NO:3, which were used as the targeting arms (homologous sequences) in the targeting construct.
  • Figure 4 shows the polynucleotide sequence for a PKL12 gene (SEQ ID NO:4).
  • Figures 5 and 6 show the design of the targeting construct used to disrupt a PKL12 gene, as well as the location and extent of the deletion in the target sequence.
  • Figure 6 shows the sequences identified as SEQ ID NO:5 and SEQ ID NO:6, which were used as the targeting arms (homologous sequences) in the targeting construct.
  • the invention is based, in part, on the evaluation of the expression and role of genes and gene expression products, primarily those associated with a target gene.
  • the invention permits the definition of disease pathways and the identification of diagnostically and therapeutically useful targets.
  • genes which are mutated or down-regulated under disease conditions may be involved in causing or exacerbating the disease condition.
  • Treatments directed at up- regulating the activity of such genes or treatments which involve alternate pathways, may ameliorate the disease condition.
  • the targeting construct of the present invention may be produced using standard methods known in the art. (See, e.g., Sambrook, et al, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; E.N. Glover (eds.), 1985, DNA Cloning: A Practical Approach, Volumes I and II; MJ. Gait (ed.), 1984, Oligonucleotide Synthesis; B.D. Hames & S.J. Higgins (eds.), 1985, Nucleic Acid Hybridization; B.D. Hames & S.J. Higgins (eds.), 1984, Transcription and Translation; R.I.
  • the targeting construct may be prepared in accordance with conventional ways, where sequences may be synthesized, isolated from natural sources, manipulated, cloned, ligated, subjected to in vitro mutagenesis, primer repair, or the like. At various stages, the joined sequences may be cloned, and analyzed by restriction analysis, sequencing, or the like.
  • the targeting DNA can be constructed using techniques well known in the art.
  • the targeting DNA may be produced by chemical synthesis of oligonucleotides, nick-translation of a double-stranded DNA template, polymerase chain-reaction amplification of a sequence (or ligase chain reaction amplification), purification of prokaryotic or target cloning vectors harboring a sequence of interest (e.g., a cloned cDNA or genomic DNA, synthetic DNA or from any of the aforementioned combination) such as plasmids, phagemids, YACs, cosmids, bacteriophage DNA, other viral DNA or replication intermediates, or purified restriction fragments thereof, as well as other sources of single and double-stranded polynucleotides having a desired nucleotide sequence.
  • the length of homology may be selected using known methods in the art. For example, selection may be based on the sequence composition and complexity of the predetermined endogenous target DNA sequence(s
  • the targeting construct of the present invention typically comprises a first sequence homologous to a portion or region of the target gene and a second sequence homologous to a second portion or region of the target gene.
  • the targeting construct further comprises a positive selection marker, which is preferably positioned in between the first and the second DNA sequence that are homologous to a portion or region of the target DNA sequence.
  • the positive selection marker may be operatively linked to a promoter and a polyadenylation signal.
  • the targeting construct may also include a sequence coding for a screening marker, for example, green fluorescent protein (GFP), or another modified fluorescent protein.
  • GFP green fluorescent protein
  • each fragment is greater than about 1 kb in length, more preferably between about 1 and about 10 kb, and even more preferably between about 1 and about 5 kb.
  • larger fragments may increase the number of homologous recombination events in ES cells, larger fragments will also be more difficult to clone.
  • the targeting construct is prepared directly from a plasmid genomic library using the methods described in pending U.S. Patent Application No.: 08/971,310, filed November 17, 1997, the disclosure of which is incorporated herein in its entirety.
  • a sequence of interest is identified and isolated from a plasmid library in a single step using, for example, long-range PCR. Following isolation of this sequence, a second polynucleotide that will disrupt the target sequence can be readily inserted between two regions encoding the sequence of interest.
  • the construct is generated in two steps by (1) amplifying (for example, using long-range PCR) sequences homologous to the target sequence, and (2) inserting another polynucleotide (for example a selectable marker) into the PCR product so that it is flanked by the homologous sequences.
  • the vector is a plasmid from a plasmid genomic library.
  • the completed construct is also typically a circular plasmid.
  • the targeting construct is designed in accordance with the regulated positive selection method described in U.S. Application No. 60/232,957, filed September 15, 2000, the disclosure of which is incorporated herein in its entirety.
  • the targeting construct is designed to include a PGK-ne ⁇ fusion gene having two lacO sites, positioned in the PGK promoter and an NLS- lacl gene comprising a lac repressor fused to sequences encoding the NLS from the SV40 T antigen.
  • the targeting construct may contain more than one selectable maker gene, including a negative selectable marker, such as the herpes simplex virus tk (HSV-tk) gene.
  • a negative selectable marker such as the herpes simplex virus tk (HSV-tk) gene.
  • the negative selectable marker may be operatively linked to a promoter and a polyadenylation signal.
  • Patent No. 5,631,153 discloses a patent No.
  • the targeting construct may be introduced into an appropriate host cell using any method known in the art.
  • Various techniques may be employed in the present invention, including, for example, pronuclear microinjection; retrovirus mediated gene transfer into germ lines; gene targeting in embryonic stem cells; electroporation of embryos; sperm-mediated gene transfer; and calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like (See, e.g., U.S. Pat. No.
  • the targeting construct is introduced into host cells by electroporation.
  • electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the construct.
  • the pores created during electroporation permit the uptake of macromolecules such as DNA.
  • Any cell type capable of homologous recombination may be used in the practice of the present invention.
  • target cells include cells derived from vertebrates including mammals such as humans, bovine species, ovine species, murine species, simian species, and ether eucaryotic organisms such as filamentous fungi, and higher multicellular organisms such as plants.
  • ES cells include embryonic stem (ES) cells, which are typically obtained from pre- implantation embryos cultured in vitro.
  • ES embryonic stem
  • the ES cells are cultured and prepared for introduction of the targeting construct using methods well known to the skilled artisan. (See, e.g., Robertson, E. J. ed.
  • the ES cells that will be inserted with the targeting construct are derived from an embryo or blastocyst of the same species as the developing embryo into which they are to be introduced. ES cells are typically selected for their ability to integrate into the inner cell mass and contribute to the germ line of an individual when introduced into the mammal in an embryo at the blastocyst stage of development. Thus, any ES cell line having this capability is suitable for use in the practice of the present invention.
  • the present invention may also be used to knockout genes in other cell types, such as stem cells.
  • stem cells may be myeloid, lymphoid, or neural progenitor and precursor cells. These cells comprising a disruption or knockout of a gene may be particularly useful in the study of target gene function in individual developmental pathways.
  • Stem cells may be derived from any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-human primates and the like.
  • the targeting construct After the targeting construct has been introduced into cells, the cells where successful gene targeting has occurred are identified. Insertion of the targeting construct into the targeted gene is typically detected by identifying cells for expression of the marker gene.
  • the cells transformed with the targeting construct of the present invention are subjected to treatment with an appropriate agent that selects against cells not expressing the selectable marker. Only those cells expressing the selectable marker gene survive and/or grow under certain conditions. For example, cells that express the introduced neomycin resistance gene are resistant to the compound G418, while cells that do not express the neo gene marker are killed by G418.
  • the targeting construct also comprises a screening marker such as GFP
  • a screening marker such as GFP
  • homologous recombination can be identified through screening cell colonies under a fluorescent light. Cells that have undergone homologous recombination will have deleted the GFP gene and will not fluoresce.
  • the targeting construct is designed so that the expression of the selectable marker gene is regulated in a manner such that expression is inhibited following random integration but is permitted (derepressed) following homologous recombination. More particularly, the transfected cells are screened for expression of the neo gene, which requires that (1) the cell was successfully electroporated, and (2) lac repressor inhibition of neo transcription was relieved by homologous recombination. This method allows for the identification of transfected cells and homologous recom- binants to occur in one step with the addition of a single drug.
  • a positive-negative selection technique may be used to select homologous recombinants.
  • This technique involves a process in which a first drug is added to the cell population, for example, a neomycin-like drug to select for growth of transfected cells, i.e. positive selection.
  • a second drug, such as FIAU is subsequently added to kill cells that express the negative selection marker, i.e. negative selection.
  • Cells that contain and express the negative selection marker are killed by a selecting agent, whereas cells that do not contain and express the negative selection marker survive.
  • cells with non-homologous insertion of the construct express HSV thymidine kinase and therefore are sensitive to the herpes drugs such as gancyclovir (GANG) or FIAU (l-(2- deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil).
  • GANG gancyclovir
  • FIAU l-(2- deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil.
  • Successful recombination may be identified by analyzing the DNA of the selected cells to confirm homologous recombination.
  • Various techniques known in the art such as PCR and/or Southern analysis may be used to confirm homologous recombination events.
  • Homologous recombination may also be used to disrupt genes in stem cells, and other cell types, which are not totipotent embryonic stem cells.
  • stem cells may be myeloid, lymphoid, or neural progenitor and precursor cells.
  • Such transgenic cells may be particularly useful in the study of gene function in individual developmental pathways.
  • Stem cells may be derived from any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-human primates and the like.
  • cells which are not totipotent it may be desirable to knock out both copies of the target using methods which are known in the art.
  • a positive selection marker e.g., Neo r
  • cells comprising homologous recombination at a target locus which have been selected for expression of a positive selection marker (e.g., Neo r ) and screened for non-random integration can be further selected for multiple copies of the selectable marker gene by exposure to elevated levels of the selective agent (e.g., G418).
  • the cells are then analyzed for homozygosity at the target locus.
  • a second construct can be generated with a different positive selection marker inserted between the two homologous sequences.
  • the two constructs can be introduced into the cell either sequentially or simultaneously, followed by appropriate selection for each of the positive marker genes.
  • the final cell is screened for homologous recombination of both alleles of the target.
  • Selected cells are then injected into a blastocyst (or other stage of development suitable for the purposes of creating a viable animal, such as, for example, a morula) of an animal (e.g., a mouse) to form chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152 (1987)).
  • selected ES cells can be allowed to aggregate with dissociated mouse embryo cells to form the aggregation chimera.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Chimeric progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • chimeric progeny mice are used to generate a mouse with a heterozygous disruption in the gene. Heterozygous transgenic mice can then be mated. It is well know in the art that typically of the offspring of such matings will have a homozygous disruption in the target gene. The heterozygous and homozygous transgenic mice can then be compared to normal, wild type mice to determine whether disruption of the target gene causes phenotypic changes, especially pathological changes.
  • heterozygous and homozygous mice may be evaluated for phenotypic changes by physical examination, necropsy, histology, clinical chemistry, complete blood count, body weight, organ weights, and cytological evaluation of bone marrow.
  • the phenotype (or phenotypic change) associated with a disruption in the target gene is placed into or stored in a database.
  • the database includes: (i) genotypic data (e.g., identification of the disrupted gene) and (ii) phenotypic data (e.g., phenotype(s) resulting from the gene disruption) associated with the genotypic data.
  • the database is preferably electronic.
  • conditional transgenic Animals The present invention further contemplates conditional transgenic or knockout animals, such as those produced using recombination methods.
  • Bacteriophage PI Cre recombinase and flp recombinase from yeast plasmids are two non-limiting examples of site-specific DNA recombinase enzymes which cleave DNA at specific target sites (lox P sites for cre recombinase and frt sites for flp recombinase) and catalyze a ligation of this DNA to a second cleaved site.
  • recombinases include the Int recombinase of bacteriophage ⁇ (with or without Xis) (Weisberg, R. et. al., in Lambda II, (Hendrix, R, et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 211-50 (1983), herein incorporated by reference); Tpnl and the ⁇ -lactamase transposons (Mercier, et al, J.
  • Cre has been purified to homogeneity, and its reaction with the loxP site has been extensively characterized (Abremski & Hess /. Mol. Biol. 259:1509-14 (1984), herein incorporated by reference). Cre protein has a molecular weight of 35,000 and can be obtained commercially from New England Nuclear/DuPont. The cre gene (which encodes the Cre protein) has been cloned and expressed (Abremski, et al. Cell 32: 1301-11 (1983), herein incorporated by reference). The Cre protein mediates recombination between two loxP sequences (Sternberg, et al. Cold Spring Harbor Symp. Quant. Biol.
  • a circular DNA molecule having two loxP sites in direct orientation will recombine to produce two smaller circles, whereas circular molecules having two loxP sites in an inverted orientation simply invert the DNA sequences flanked by the loxP sites.
  • recombinase action can result in reciprocal exchange of regions distal to the target site when targets are present on separate DNA molecules.
  • Recombinases have important application for characterizing gene function in knockout models.
  • a fusion transcript can be produced when insertion of the positive selection marker occurs downstream (3') of the translation initiation site of the target gene.
  • the fusion transcript could result in some level of protein expression with unknown consequence. It has been suggested that insertion of a positive selection marker gene can affect the expression of nearby genes. These effects may make it difficult to determine gene function after a knockout event since one could not discern whether a given phenotype is associated with the inactivation of a gene, or the transcription of nearby genes. Both potential problems are solved by exploiting recombinase activity.
  • purified recombinase enzyme is provided to the cell by direct microinjection.
  • recombinase is expressed from a co-transfected construct or vector in which the recombinase gene is operably linked to a functional promoter.
  • tissue-specific or inducible recombinase constructs which allow the choice of when and where recombination occurs.
  • One method for practicing the inducible forms of recombinase-mediated recombination involves the use of vectors that use inducible or tissue- specific promoters or other gene regulatory elements to express the desired recombinase activity.
  • the inducible expression elements are preferably operatively positioned to allow the inducible control or activation of expression of the desired recombinase activity.
  • Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (No, et al. Proc. Natl. Acad. Sci.
  • control elements that can be used include promoters requiring specific transcription factors such as viral, promoters. Vectors incorporating such promoters would only express recombinase activity in cells that express the necessary transcription factors.
  • the cell- and animal-based systems described herein can be utilized as models for diseases.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate disease animal models.
  • cells from humans may be used.
  • Such assays may be utilized as part of screening strategies designed to identify agents, such as compounds which are capable of ameliorating disease symptoms.
  • the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease.
  • Cell-based systems may be used to identify compounds which may act to ameliorate disease symptoms. For example, such cell systems may be exposed to a compound suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed cells. After exposure, the cells are examined to determine whether one or more of the disease cellular phenotypes has been altered to resemble a more normal or more wild type, non-disease phenotype.
  • animal-based disease systems such as those described herein, may be used to identify compounds capable of ameliorating disease symptoms.
  • animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating a disease or other phenotypic characteristic of the animal.
  • animal models may be exposed to a compound or agent suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed animals.
  • the response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with the disease. Exposure may involve treating mother animals during gestation of the model animals described herein, thereby exposing embryos or fetuses to the compound or agent which may prevent or ameliorate the disease or phenotype. Neonatal, juvenile, and adult animals can also be exposed.
  • the present invention provides a method of identifying agents having an effect on target gene or alternatively, target protein expression or function.
  • the method includes measuring a physiological response of the animal, for example, to the agent, and comparing the physiological response of such animal to a control animal, wherein the physiological response of the animal comprising a disruption in a target gene as compared to the control animal indicates the specificity of the agent.
  • a "physiolo- gical response" is any biological or physical parameter of an animal which can be measured.
  • Molecular assays e.g., gene transcription, protein production and degradation rates
  • physical parameters e.g., exercise physiology tests, measurement of various parameters of respiration, measurement of heart rate or blood pressure, measurement of bleeding time, aPTT.T, or TT
  • cellular assays e.g.,. immunohistochemical assays of cell surface markers, or the ability of cells to aggregate or proliferate
  • the transgenic animals and cells of the present invention may by utilized as models for diseases, disorders, or conditions associated with phenotypes relating to a disruption in a target gene.
  • the present invention provides a unique animal model for testing and developing new treatments relating to the behavioral phenotypes. Analysis of the behavioral phenotype allows for the development of an animal model useful for testing, for instance, the efficacy of proposed genetic and pharmacological therapies for human genetic diseases, such as neurological, neuropsychological, or psychotic illnesses.
  • a statistical analysis of the various behaviors measured can be carried out using any conventional statistical program routinely used by those skilled in the art (such as, for example, "Analysis of Variance” or ANOVA).
  • a "p" value of about 0.05 or less is generally considered to be statistically significant, although slightly higher p values may still be indicative of statistically significant differences.
  • a comparison is made between the behavior of a transgenic animal (or a group thereof) to the behavior of a wild-type mouse (or a group thereof), typically under certain prescribed conditions.
  • “Abnormal behavior” as used herein refers to behavior exhibited by an animal having a disruption in the target gene, e.g. transgenic animal, which differs from an animal without a disruption in the target gene, e.g.
  • Abnormal behavior consists of any number of standard behaviors that can be objectively measured (or observed) and compared. In the case of comparison, it is preferred that the change be statistically significant to confirm that there is indeed a meaningful behavioral difference between the knockout animal and the wild-type control animal.
  • behaviors which may be measured or observed include, but are not limited to, ataxia, rapid limb movement, eye movement, breathing, motor activity, cognition, emotional behaviors, social behaviors, hyperactivity, hypersensitivity, anxiety, impaired learning, abnormal reward behavior, and abnormal social interaction, such as aggression.
  • a series of tests may be used to measure the behavioral phenotype of the animal models of the present invention, including neurological and neuropsychological tests to identify abnormal behavior. These tests may be used to measure abnormal behavior relating to, for example, learning and memory, eating, pain, aggression, sexual reproduction, anxiety, depression, schizophrenia, and drug abuse. (See, e.g., Crawley and Paylor, Hormones and Behavior 31:197-211 (1997)).
  • the social interaction test involves exposing a mouse to other animals in a variety of settings.
  • the social behaviors of the animals e.g., touching, climbing, sniffing, and mating
  • Differences in behaviors can then be statistically analyzed and compared (See, e.g., S. E. File, et al, Pharmacol. Bioch. Behav. 22:941-944 (1985); R. R. Holson, Phys. Behav. 37:239-247 (1986)).
  • Examplary behavioral tests include the following.
  • the mouse startle response test typically involves exposing the animal to a sensory (typically auditory) stimulus and measuring the startle response of the animal (see, e.g., M. A. Geyer, et al, Brain Res. Bull. 25:485-498 (1990); Paylor and Crawley, Psychopharmacology 132:169-180 (1997)).
  • a pre-pulse inhibition test can also be used, in which the percent inhibition (from a normal startle response) is measured by "cueing" the animal first with a brief low-intensity pre-pulse prior to the startle pulse.
  • the electric shock test generally involves exposure to an electrified surface and measurement of subsequent behaviors such as, for example, motor activity, learning, social behaviors.
  • the behaviors are measured and statistically analyzed using standard statistical tests. (See, e.g., G. J. Kant, et al, Pharm. Bioch. Behav. 20:793-797 (1984); N. J. Leidenheimer, et al, Pharmacol. Bioch. Behav. 30:351-355 (1988)).
  • the tail-pinch or immobilization test involves applying pressure to the tail of the animal and/or restraining the animal's movements.
  • Motor activity, social behavior, and cognitive behavior are examples of the areas that are measured. (See, e.g., M. Bertolucci D'Angic, et al, Neurochem. 55:1208-1214 (1990)).
  • the novelty test generally comprises exposure to a novel environment and/or novel objects.
  • the animal's motor behavior in the novel environment and/or around the novel object are measured and statistically analyzed.
  • This test may be used to detect visual processing deficiencies or defects.
  • the learned helplessness test involves exposure to stresses, for example, noxious stimuli, which cannot be affected by the animal's behavior.
  • the animal's behavior can be statistically analyzed using various standard statistical tests. (See, e.g., A. Leshner, et al, Behav. Neural Biol. 26:497-501 (1979)).
  • a tail suspension test may be used, in which the "immobile" time of the mouse is measured when suspended “upside-down” by its tail. This is a measure of whether the animal struggles, an indicator of depression.
  • depression is believed to result from feelings of a lack of control over one's life or situation. It is believed that a depressive state can be elicited in animals by repeatedly subjecting them to aversive situations over which they have no control. A condition of "learned helplessness" is eventually reached, in which the animal will stop trying to change its circumstances and simply accept its fate. Animals that stop struggling sooner are believed to be more prone to depression. Studies have shown that the administration of certain antidepressant drugs prior to testing increases the amount of time that animals struggle before giving up.
  • the Morris water-maze test comprises learning spatial orientations in water and subsequently measuring the animal's behaviors, such as, for example, by counting the number of incorrect choices.
  • the behaviors measured are statistically analyzed using standard statistical tests.
  • a Y-shaped maze may be used (see, e.g., McFarland, D.J., Pharmacology,
  • the Y-maze is generally believed to be a test of cognitive ability.
  • the dimensions of each arm of the Y-maze can be, for example, approximately 40 cm x 8 cm x 20 cm, although other dimensions may be used.
  • Each arm can also have, for example, sixteen equally spaced photobeams to automatically detect movement within the arms. At least two different tests can be performed using such a Y-maze. In a continuous Y-maze paradigm, mice are allowed to explore all three arms of a Y-maze for, e.g., approximately 10 minutes.
  • the animals are continuously tracked using photobeam detection grids, and the data can be used to measure spontaneous alteration and positive bias behavior.
  • Spontaneous alteration refers to the natural tendency of a "normal" animal to visit the least familiar arm of a maze.
  • An alternation is scored when the animal makes two consecutive turns in the same direction, thus representing a sequence of visits to the least recently entered arm of the maze.
  • Position bias determines egocentrically defined responses by measuring the animal's tendency to favor turning in one direction over another. Therefore, the test can detect differences in an animal's ability to navigate on the basis of allocentric or egocentric mechanisms.
  • the two-trial Y-maze memory test measures response to novelty and spatial memory based on a free- choice exploration paradigm.
  • the animals are allowed to freely visit two arms of the Y-maze for, e.g., approximately 15 minutes.
  • the third arm is blocked off during this trial.
  • the second trial (retrieval) is performed after an intertrial interval of, e.g., approximately 2 hours.
  • the blocked arm is opened and the animal is allowed access to all three arms for, e.g., approximately 5 minutes.
  • Data are collected during the retrieval trial and analyzed for the number and duration of visits to each arm. Because the three arms of the maze are virtually identical, discrimination between novelty and familiarity is dependent on "environmental" spatial cues around the room relative to the position of each arm. Changes in arm entry and duration of time spent in the novel arm in a transgenic animal model may be indicative of a role of that gene in mediating novelty and recognition processes.
  • the passive avoidance or shuttle box test generally involves exposure to two or more environments, one of which is noxious, providing a choice to be learned by the animal. Behavioral measures include, for example, response latency, number of correct responses, and consistency of response. (See, e.g., R. Ader, et al, Psychon. Sci. 26:125-128 (1972); R. R. Holson, Phys. Behav. 37:221-230 (1986)).
  • a zero-maze can be used.
  • the animals can, for example, be placed in a closed quadrant of an elevated annular platform having, e.g., 2 open and 2 closed quadrants, and are allowed to explore for approximately 5 minutes.
  • This paradigm exploits an approach-avoidance conflict between normal exploratory activity and an aversion to open spaces in rodents.
  • This test measures anxiety levels and can be used to evaluate the effectiveness of anti- anxiolytic drugs.
  • the time spent in open quadrants versus closed quadrants may be recorded automatically, with, for example, the placement of photobeams at each transition site.
  • the food avoidance test involves exposure to novel food and objectively measuring, for example, food intake and intake latency.
  • the behaviors measured are statistically analyzed using standard statistical tests.
  • the elevated plus-maze test comprises exposure to a maze, without sides, on a platform, the animal's behavior is objectively measured by counting the number of maze entries and maze learning.
  • the behavior is statistically analyzed using standard statistical tests. (See, e.g., H. A. Baldwin, et al, Brain Res. Bull, 20:603-606 (1988)).
  • the stimulant-induced hyperactivity test involves injection of stimulant drugs (e.g., amphetamines, cocaine, PCP, and the like), and objectively measuring, for example, motor activity, social interactions, cognitive behavior.
  • stimulant drugs e.g., amphetamines, cocaine, PCP, and the like
  • the animal's behaviors are statistically analyzed using standard statistical tests. (See, e.g., P. B. S. Clarke, et al, Psychopharmacology 96:511-520 (1988); P.
  • the self-stimulation test generally comprises providing the mouse with the opportunity to regulate electrical and/or chemical stimuli to its own brain. Behavior is measured by frequency and pattern of self-stimulation. Such behaviors are statistically analyzed using standard statistical tests.
  • the reward test involves shaping a variety of behaviors, e.g., motor, cognitive, and social, measuring, for example, rapidity and reliability of behavioral change, and statistically analyzing the behaviors measured.
  • behaviors e.g., motor, cognitive, and social
  • the DRL (differential reinforcement to low rates of responding) performance test involves exposure to intermittent reward paradigms and measuring the number of proper responses, e.g., lever pressing. Such behavior is statistically analyzed using standard statistical tests. (See, e.g., J. D. Sinden, et al, Behav. Neuroscl 100:320-329 (1986); V. Nalwa, et al, Behav Brain Res. 17:73-76 (1985); and
  • the spatial learning test involves exposure to a complex novel environment, measuring the rapidity and extent of spatial learning, and statistically analyzing the behaviors measured. (See, e.g.,
  • an open-field (of) test may be used, in which the greater distance traveled for a given amount of time is a measure of the activity level and anxiety of the animal.
  • an open field is a novel environment, it is believed that an approach-avoidance situation is created, in which the animal is "torn" between the drive to explore and the drive to protect itself. Because the chamber is lighted and has no places to hide other than the corners, it is expected that a "normal” mouse will spend more time in the corners and around the periphery than it will in the center where there is no place to hide. "Normal" mice will, however, venture into the central regions as they explore more and more of the chamber.
  • mice will spend most of their time in the corners, with relatively little or no exploration of the central region, whereas bold (i.e., less anxious) mice will travel a greater distance, showing little preference for the periphery versus the central region.
  • the visual, somatosensory and auditory neglect tests generally comprise exposure to a sensory stimulus, objectively measuring, for example, orientating responses, and statistically analyzing the behaviors measured. (See, e.g., J. M. Vargo, et al., Exp. Neurol. 102:199-209 (1988)).
  • the eonsummatory behavior test generally comprises feeding and drinking, and objectively measuring quantity of consumption.
  • the behavior measured is statistically analyzed using standard statistical tests. (See, e.g., P. J. Fletcher, et al., Psychopharmacol. 102:301-308 (1990); M. G. Corda, et al.,, Proc. Nat Acad. Sci. USA 80:2072-2076 (1983)).
  • a visual discrimination test can also be used to evaluate the visual processing of an animal.
  • One or two similar objects are placed in an open field and the animal is allowed to explore for about 5-10 minutes.
  • the time spent exploring each object proximity to, i.e., movement within, e.g., about 3-5 cm of the object is considered exploration of an object
  • the animal is then removed from the open field, and the objects are replaced by a similar object and a novel object.
  • the animal is returned to the open field and the percent time spent exploring the novel object over the old object is measured (again, over about a 5-10 minute span). "Normal" animals will typically spend a higher percentage of time exploring the novel object rather than the old object.
  • the memory task becomes more hippocampal-dependent. If no delay is imposed, the task is more based on simple visual discrimination.
  • This test can also be used for olfactory discrimination, in which the objects (preferably, simple blocks) can be sprayed or otherwise treated to hold an odor. This test can also be used to determine if the animal can make gustatory discriminations; animals that return to the previously eaten food instead of novel food exhibit gustatory neophobia.
  • a hot plate analgesia test can be used to evaluate an animal's sensitivity to heat or painful stimuli. For example, a mouse can be placed on an approximately 55°C hot plate and the mouse's response latency (e.g., time to pick up and lick a hind paw) can be recorded. These responses are not reflexes, but rather "higher" responses requiring cortical involvement. This test may be used to evaluate a nociceptive disorder.
  • An accelerating rotarod test may be used to measure coordination and balance in mice. Animals can be, for example, placed on a rod that acts like a rotating treadmill (or rolling log).
  • the rotarod can be made to rotate slowly at first and then progressively faster until it reaches a speed of, e.g., approximately 60 rpm.
  • the mice must continually reposition themselves in order to avoid falling off.
  • the animals are preferably tested in at least three trials, a minimum of 20 minutes apart. Those mice that are able to stay on the rod the longest are believed to have better coordination and balance.
  • a metrazol administration test can be used to screen animals for varying susceptibilities to seizures or similar events.
  • a 5mg/ml solution of metrazol can be infused through the tail vein of a mouse at a rate of, e.g., approximately 0.375 ml/min.
  • the infusion will cause all mice to experience seizures, followed by death.
  • Those mice that enter the seizure stage the soonest are believed to be more prone to seizures.
  • Four distinct physiological stages can be recorded: soon after the start of infusion, the mice will exhibit a noticeable "twitch", followed by a series of seizures, ending in a final tensing of the body known as "tonic extension", which is followed by death.
  • Target Gene Products may include proteins that represent functionally equivalent gene products.
  • Such an equivalent gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the gene sequences described herein, but which result in a silent change, thus producing a functionally equivalent target gene product.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobi- city, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Functionally equivalent”, as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous gene products encoded by the target gene sequences.
  • “functionally equivalent” may refer to peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous gene product would.
  • Protein products useful according to the methods of the invention are peptides derived from or based on the target gene produced by recombinant or synthetic means (derived peptides).
  • Target gene products may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing the gene polypeptides and peptides of the invention by expressing nucleic acid encoding gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing gene protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination (see, e.g., Sambrook, et al., 1989, supra, and Ausubel, et al., 1989, supra).
  • RNA capable of encoding gene protein sequences may be chemically synthesized using, for example, automated synthesizers (see, e.g. Oligonucleotide Synthesis: A Practical Approach, Gait, M. J. ed., IRL Press, Oxford (1984)).
  • host-expression vector systems may be utilized to express the gene coding sequences of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the gene protein of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing gene protein coding sequences; yeast (e.g.
  • Saccharomyces, Pichia transformed with recombinant yeast expression vectors containing the gene protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing gene protein coding sequences; or mammalian cell systems (e.g.
  • COS COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter).
  • promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the geiie protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E.
  • coli expression vector pUR278 (Ruther et al, EMBO /., 2: 1791-94 (1983)), in which the gene protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Jnouye, Nucleic Acids Res., 13:3101-09 (1985); Van Heeke et al, J. Biol. Chem., 264:5503-9 (1989)); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
  • full length cDNA sequences are appended with in-frame Bam HI sites at the amino terminus and Eco Rl sites at the carboxyl terminus using standard PCR methodo- logies (Innis, et al. (eds) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990)) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala, Sweden).
  • the resulting cDNA construct contains a kinase recognition site at the amino terminus for radioactive labeling and glutathione S-transferase sequences at the carboxyl terminus for affinity purification (Nilsson, et al, EMBO J., 4: 1075-80 (1985); Zabeau et al, EMBO J., 1: 1217-24 (1982)).
  • AdNPV Autographa calif ornica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (see, e.g., Smith, et al, J. Virol 46: 584-93 (1983); U.S. Pat. No. 4,745,051).
  • a number of viral-based expression systems may be utilized.
  • the gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing gene protein in infected hosts, (e.g., see Logan et al, Proc. Natl. Acad. Sci.
  • Specific initiation signals may also be required for efficient translation of inserted gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter, et al, Methods in EnzymoL, 153:516-44 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechan- isms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
  • cell lines which stably express the gene protein may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells which stably integrate the plasmid into their chromosomes and grow, to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the gene protein.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the gene protein.
  • control of timing and/or quantity of expression of the recombinant protein can be controlled using an inducible expression construct.
  • inducible constructs and systems for inducible expression of recombinant proteins will be well known to those skilled in the art.
  • examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (No, et al, Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al, Proc. Natl. Acad. Sci.
  • Tet inducible gene expression system is utilized. (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-51 (1992); Gossen, et al, Science, 268:1766-69 (1995)). Tet Expression Systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E.
  • TetR tetracycline repressor protein
  • tetO tetracycline operator sequence
  • expression of the recombinant protein is placed under the control of the tetO operator sequence and transfected or transformed into a host cell.
  • TetR which is co-transfected into the host cell
  • expression of the recombinant protein is repressed due to binding of the TetR protein to the tetO regulatory element.
  • Tc tetracycline
  • Dox doxycycline
  • the gene protein When used as a component in an assay system, the gene protein may be labeled, either directly or indirectly, to facilitate detection of a complex formed between the gene protein and a test substance.
  • labeling systems Any of a variety of suitable labeling systems may be used including but not limited to radioisotopes such as 1251; enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to substrate; and fluorescent labels.
  • radioisotopes such as 1251
  • enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to substrate
  • fluorescent labels Where recombinant DNA technology is used to produce the gene protein for such assay systems, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization and/or detection.
  • Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to the gene product.
  • a protein such as a labeled antibody
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of a target gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal target gene activity.
  • such antibodies may be utilized as part of disease treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of target gene proteins, or for the presence of abnormal forms of such proteins.
  • various host animals may be immunized by injection with the target gene, its expression product or a portion thereof.
  • Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ⁇ ) and Corynebacterium parvum.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
  • an antigen such as target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection with gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of K ⁇ hler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor, et al, Immunology Today, 4:72 (1983); Cote, et al, Proc. Natl Acad. Sci. USA, 80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al, in Monoclonal Antibodies And Cancer Therapy, Alan R.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al, Proc. Natl. Acad.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed (Huse, et al, Science, 246: 1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Screening Methods The present invention may be employed in a process for screening for agents such as agonists, i.e.
  • polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures as known in the art. Any methods routinely used to identify and screen for agents that can modulate receptors may be used in accordance with the present invention.
  • the present invention provides methods for identifying and screening for agents that modulate target gene expression or function. More particularly, cells that contain and express target gene sequences may be used to screen for therapeutic agents. Such cells may include non- recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TTB-202), and P388D1 (ATCC# TU3-63); endothelial cells such as HUVEC's and bovine aortic endothelial cells (BAEC's); as well as generic mammalian cell lines such as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL-1651). Further, such cells may include recombinant, transgenic cell lines.
  • monocyte cell lines such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TTB-202), and P388D1 (ATCC# TU3-63)
  • endothelial cells such as HUVEC's and bovine aortic
  • the transgenic mice of the invention may be used to generate cell lines, containing one or more cell types involved in a disease, that can be used as cell culture models for that disorder. While cells, tissues, and primary cultures derived from the disease transgenic animals of the invention may be utilized, the generation of continuous cell lines is preferred. For examples of techniques which may be used to derive a continuous cell line from the transgenic animals, see Small, et al., Mol. Cell Biol, 5:642-48 (1985).
  • Target gene sequences may be introduced into, and overexpressed in, the genome of the cell of interest.
  • the coding portion of the target gene sequence may be ligated to a regulatory sequence which is capable of driving gene expression in the cell type of interest.
  • a regulatory sequence which is capable of driving gene expression in the cell type of interest.
  • Such regulatory regions will be well known to those of skill in the art, and may be utilized in the absence of undue experimentation.
  • Target gene sequences may also be disrupted or underexpressed. Cells having target gene disruptions or underexpressed target gene sequences may be used, for example, to screen for agents capable of affecting alternative pathways which compensate for any loss of function attributable to the disruption or underexpression.
  • In vitro systems may be designed to identify compounds capable of binding the target gene products.
  • Such compounds may include, but are not limited to, peptides made of D-and/or L-configu- ration amino acids (in, for example, the form of random peptide libraries; see e.g., Lam, et al, Nature, 354:82-4 (1991)), phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al, Cell, 72:767-78 (1993)), antibodies, and small organic or inorganic molecules.
  • Target gene proteins preferably mutant target gene proteins
  • elaborating the biological function of the target gene protein or screening for compounds that disrupt normal target gene interactions or themselves disrupt such interactions.
  • the principle of the assays used to identify compounds that bind to the target gene protein involves preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.
  • one method to conduct such an assay would involve anchoring the target gene protein or the test substance onto a solid phase and detecting target protein/ test substance complexes anchored on the solid phase at the end of the reaction.
  • the target gene protein may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
  • microtitre plates are conveniently utilized.
  • the anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein may be used to anchor the protein to the solid surface.
  • the surfaces may be prepared in advance and stored.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • agents which may be used as therapeutics include the target gene, its expression product(s) and functional fragments thereof. Additionally, agents which reduce or inhibit mutant target gene activity may be used to ameliorate disease symptoms. Such agents include antisense, ribozyme, and triple helix molecules. Techniques for the production and use of such molecules are well known to those of skill in the art.
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest, are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
  • RNA sequences encoding target gene proteins are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxyribonucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.-
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the antisense, ribozyme, and/or triple helix molecules described herein may reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by both normal and mutant target gene alleles.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal activity may be introduced into cells that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • Various well-known modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • Antibodies that are both specific for target gene protein, and in particular, mutant gene protein, and interfere with its activity may be used to inhibit mutant target gene function.
  • Such antibodies may be generated against the proteins themselves or against peptides corresponding to portions of the proteins using standard techniques known in the art and as also described herein.
  • Such antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, etc.
  • internalizing antibodies may be preferred.
  • lipofectin liposomes may be used to deliver the antibody or a fragment of the Fab region which binds to the target gene epitope into cells.
  • fragments of the antibody are used, the smallest inhibitory fragment which binds to the target or expanded target protein's binding domain is preferred.
  • peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used.
  • Such peptides may be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (see, e.g., Creighton, Proteins: Structures and Molecular Principles (1984) W.H. Freeman, New York 1983, supra; and Sambrook, et al, 1989, supra).
  • single chain neutralizing antibodies which bind to intracellular target gene epitopes may also be administered.
  • Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco, et al., Proc. Natl. Acad. Sci. USA, 90:7889-93 (1993).
  • RNA sequences encoding target gene protein may be directly administered to a patient exhibiting disease symptoms, at a concentration sufficient to produce a level of target gene protein such that disease symptoms are ameliorated. Patients may be treated by gene replacement therapy.
  • One or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein with target gene function may be inserted into cells using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Additionally, techniques such as those described above may be utilized for the introduction of normal target gene sequences into human cells.
  • Cells preferably, autologous cells, containing normal target gene expressing gene sequences may then be introduced or reintroduced into the patient at positions which allow for the amelioration of disease symptoms.
  • compositions comprising: Compositions, Effective Dosages, and Routes of Administration
  • the identified compounds that inhibit target mutant gene expression, synthesis and or activity can be administered to a patient at therapeutically effective doses to treat or ameliorate the disease.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disease.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, subcutaneous, intraperitoneal, intravenous, intrapleural, intraoccular, intraarterial, or rectal administration. It is also contemplated that pharmaceutical compositions may be administered with other products that potentiate the activity of the compound and optionally, may include other therapeutic ingredients.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methyl- cellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methyl- cellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluo
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • Oral ingestion is possibly the easiest method of taking any medication.
  • Such a route of administration is generally simple and straightforward and is frequently the least inconvenient or unpleasant route of administration from the patient's point of view.
  • this involves passing the material through the stomach, which is a hostile environment for many materials, including proteins and other biologically active compositions.
  • compositions may also include various buffers (e.g., Tris, acetate, phosphate), solubilizers (e.g., Tween, Polysorbate), carriers such as human serum albumin, preservatives (thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid in order to stabilize pharmaceu- tical activity.
  • the stabilizing agent may be a detergent, such as tween-20, tween-80, NP-40 or Triton X-100.
  • EBP may also be incorporated into particulate preparations of polymeric compounds for controlled delivery to a patient over an extended period of time. A more extensive survey of components in pharmaceutical compositions is found in Remington's Pharmaceutical Sciences, 18th ed., A. R. Gennaro, ed., Mack Publishing, Easton, Pa. (1990).
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • alteration of the wild-type target gene locus is detected.
  • the method can be performed by detecting the wild-type target gene locus and confirming the lack of a predisposition or neoplasia.
  • "Alteration of a wild-type gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline.
  • Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are mutated, then a late neoplastic state may be indicated. The finding of gene mutations thus provides both diagnostic and prognostic information.
  • a target gene allele which is not deleted e.g., that found on the sister chromosome to a chromosome carrying a target gene deletion
  • Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the target gene product, or a decrease in mRNA stability or translation efficiency.
  • One test available for detecting mutations in a candidate locus is to directly compare genomic target sequences from cancer patients with those from a control population. Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene.
  • Mutations from cancer patients falling outside the coding region of the target gene can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the target gene.
  • non-coding regions such as introns and regulatory sequences near or within the target gene.
  • An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger
  • RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific gene nucleic acid or anti-gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting disease symptoms or at risk for developing disease.
  • Any cell type or tissue preferably monocytes, endothelial cells, or smooth muscle cells, in which the gene is expressed may be utilized in the diagnostics described below.
  • DNA or RNA from the cell type or tissue to be analyzed may easily be isolated using procedures which are well known to those in the art. Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, N.Y. (1992)).
  • Gene nucleotide sequences may, for example, be used in hybridization or amplification assays of biological samples to detect disease-related gene structures and expression.
  • assays may include, but are not limited to, Southern or Northern analyses, restriction fragment length polymorphism assays, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses.
  • analyses may reveal both quantitative aspects of the expression pattern of the gene, and qualitative aspects of the gene expression and/or gene composition. That is, such aspects may include, for example, point mutations, insertions, deletions, chromosomal rearrangements, and/or activation or inactivation of gene expression.
  • Preferred diagnostic methods for the detection of gene-specific nucleic acid molecules may involve for example, contacting and incubating nucleic acids, derived from the cell type or tissue being analyzed, with one or more labeled nucleic acid reagents under conditions favorable for the specific annealing of these reagents to their complementary sequences within the nucleic acid molecule of interest.
  • the lengths of these nucleic acid reagents are at least 9 to 30 nucleotides.
  • all non-annealed nucleic acids are removed from the nucleic acid.-fingerprint molecule hybrid. The presence of nucleic acids from the fingerprint tissue which have hybridized, if any such molecules exist, is then detected.
  • the nucleic acid from the tissue or cell type of interest may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtitre plate or polystyrene beads.
  • a solid support such as a membrane, or a plastic surface such as that on a microtitre plate or polystyrene beads.
  • Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art.
  • Alternative diagnostic methods for the detection of gene-specific nucleic acid molecules may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis U.S. Pat. No. 4,683,202 (1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA, 88:189-93 (1991)), self sustained sequence replication (Guatelli, et al, Proc. Natl. Acad. Sci. USA, 87:1874-78 (1990)), transcriptional amplification system (Kwoh, et al, Proc. Natl. Acad. Sci.
  • a cDNA molecule is obtained from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA).
  • RNA types or tissues from which such RNA may be isolated include any tissue in which wild type fingerprint gene is known to be expressed, including, but not limited, to monocytes, endothelium, and/or smooth muscle.
  • a sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
  • the nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method may be chosen from among the gene nucleic acid reagents described herein.
  • the preferred lengths of such nucleic acid reagents are at least 15-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non- radioactively labeled nucleotides.
  • enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • Antibodies directed against wild type or mutant gene peptides may also be used as disease diagnostics and prognostics. Such diagnostic methods, may be used to detect abnormalities in the level of gene protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of fingerprint gene protein. Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant fingerprint gene protein relative to the normal fingerprint gene protein. Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to those of skill in the art, including but not limited to western blot analysis. For a detailed explanation of methods for carrying out western blot analysis. (See, e.g. Sambrook, et al. (1989) supra, at Chapter 18).
  • the protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)).
  • Preferred diagnostic methods for the detection of wild type or mutant gene peptide molecules may involve, for example, immunoassays wherein fingerprint gene peptides are detected by their interaction with an anti-fingerprint gene-specific peptide antibody.
  • antibodies, or fragments of antibodies useful in the present invention may be used to quantitatively or qualitatively detect the presence of wild type or mutant gene peptides. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if the fingerprint gene peptides are expressed on the cell surface.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of fingerprint gene peptides.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for wild type, mutant, or expanded fingerprint gene peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying fingerprint gene peptides, and detecting the bound antibody by any of a number of techniques well known in the art.
  • a biological sample such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled gene-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier are intended to encompass any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacryla- mides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of anti-wild type or -mutant fingerprint gene peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fingerprint gene wild type, mutant, or expanded peptides through the use of a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986).
  • RIA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • the targeting construct was introduced into ES cells by electroporation and chimeric mice were generated.
  • the ES cells were derived from 129/Sv-+P+Mgf-SLJ/J mouse substrain.
  • FI mice were generated by breeding with C57BL/6 females.
  • the resultant F1N0 hetero- zygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes.
  • F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females.
  • Example 2 Generation and Analysis of Mice Comprising PKL12 Gene Disruptions Targeting Construct.
  • disruptions in genes comprising the sequence set forth in SEQ ID NO:4 were produced by homologous recombination. More particularly, as shown in Figures 5 and 6, a specific targeting construct having the ability to disrupt or modify genes comprising SEQ ID NO:4 was created, using as the targeting arms (homologous sequences) in the construct the sequences identified herein as SEQ ID NO:5 and SEQ ID NO:6 (see Figure 6).
  • the targeting construct was introduced into ES cells derived from the 129/OlaHsd mouse substrain by electroporation to generate chimeric mice.
  • FI mice were generated by breeding with C57BL/6 females.
  • the resultant F1N0 heterozygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate FlNl heterozygotes.
  • F2N1 homozygous mutant mice were produced by intercrossing FlNl heterozygous males and females.

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Abstract

L'invention concerne des animaux transgéniques ainsi que des compositions et des méthodes se rapportant à la caractérisation de la fonction génique. D'une manière plus spécifique, l'invention concerne des souris transgéniques présentant des mutations de gène kinase cible. Lesdites souris transgéniques présentent une grande utilité comme modèles pour des maladies et permettent d'identifier des agents qui modulent l'expression et la fonction génique. En outre, l'invention concerne des traitements potentiels de diverses maladies et états pathologiques.
PCT/US2001/021821 2000-07-11 2001-07-10 Souris transgeniques contenant des interruptions de genes kinase cibles WO2002003790A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919997A (en) * 1993-11-18 1999-07-06 Cold Spring Habor Labortary Transgenic mice having modified cell-cycle regulation
WO1999066051A2 (fr) * 1998-06-16 1999-12-23 Sugen, Inc. Kinases en rapport avec nek et avec bub1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919997A (en) * 1993-11-18 1999-07-06 Cold Spring Habor Labortary Transgenic mice having modified cell-cycle regulation
WO1999066051A2 (fr) * 1998-06-16 1999-12-23 Sugen, Inc. Kinases en rapport avec nek et avec bub1

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KANDLI M ET AL: "Isolation and characterization of two evolutionarily conserved murine kinases (Nek6 and Nek7) related to the fungal mitotic regulator, NIMA" GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 68, no. 2, 1 September 2000 (2000-09-01), pages 187-196, XP002217735 ISSN: 0888-7543 *
LI M Z ET AL: "Assignment of NEK6, a NIMA-related gene, to human chromosome 9q33.3fwdarwq34.11 by radiation hybrid mapping." CYTOGENETICS AND CELL GENETICS, vol. 87, no. 3-4, 1999, pages 271-272, XP008018240 The Fifth International Workshop on Human Chromosome 1 Mapping 1999.;Cambridge, UK; August 05-07, 1999 ISSN: 0301-0171 *
LIGOS JOSE MANUEL ET AL: "Cloning, expression analysis, and functional characterization of PKL12, a member of a new subfamily of ser/thr kinases." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 249, no. 2, 19 August 1998 (1998-08-19), pages 380-384, XP002243802 ISSN: 0006-291X *

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