WO2003096804A1 - Facteur de croissance d'animal genetiquement modifie de type facteur de croissance epidermique se liant a l'heparine, procede de criblage, cellules souches embryonnaires et agent preventif et/ou remede contre l'insuffisance cardiaque - Google Patents

Facteur de croissance d'animal genetiquement modifie de type facteur de croissance epidermique se liant a l'heparine, procede de criblage, cellules souches embryonnaires et agent preventif et/ou remede contre l'insuffisance cardiaque Download PDF

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WO2003096804A1
WO2003096804A1 PCT/JP2003/005672 JP0305672W WO03096804A1 WO 2003096804 A1 WO2003096804 A1 WO 2003096804A1 JP 0305672 W JP0305672 W JP 0305672W WO 03096804 A1 WO03096804 A1 WO 03096804A1
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growth factor
egf
gene
heparin
epidermal growth
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PCT/JP2003/005672
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English (en)
Japanese (ja)
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Seiji Takashima
Masafumi Kitakaze
Eisuke Mekata
Shigeki Higashiyama
Masanori Asakura
Tohru Iwamoto
Satoru Yamazaki
Masaji Hori
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Hubit Genomix, Inc.
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Priority to AU2003235855A priority Critical patent/AU2003235855A1/en
Priority to JP2004504814A priority patent/JPWO2003096804A1/ja
Publication of WO2003096804A1 publication Critical patent/WO2003096804A1/fr

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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • 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
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • 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 a heparin-binding epidermal growth factor-like growth factor gene function-modified animal, a screening method, an embryonic stem cell, and an agent for preventing and / or treating heart failure.
  • Heparin-binding EGF-like growth factor was originally a monocyte-like cell.
  • Epidermal growth factor (EGF) isolated from U937 as a fibroblast growth factor is a growth factor belonging to the family (Higashiyama S et al. Science
  • HB-EGF Soluble HB-EGF is degraded by a protease from membrane-bound HB-EGF, released from the cell membrane and binds to epidermal growth factor receptor to exert its function. It has been reported that HB-EGF has proliferative activity on fibroblasts and smooth muscle cells and is involved in blastocyst engraftment, wound healing, tumor growth, arteriosclerosis, and the like. (For a review, see Raab G and Klagsbrun M Biochim, Biophys Acta
  • HB-EGF than cell membrane in heart It was shown that the release of EGF and stimulation of the EGF receptor is important as a signal for cardiomyocyte hypertrophy. This suggests that among the epidermal growth factor (EGF) families, HB-EGF in particular plays an important signal in cardiomyocytes (Asakura M et al. Nature Medicine 8: 35-40, 2002).
  • HB-EGF was not released from the cell membrane using a genetic modification technique.
  • a mouse was prepared in which a part of the mouse HB-EGF genomic gene was deleted and a mutant HB-EGF cDNA was introduced instead.
  • the original HB-EGF gene was broken, its transcript was not expressed, and only the mutant HB-EGF was expressed.
  • the mouse of this strain was named HB-EGF pr . / pr . Write mouse.
  • HB- EGF p r ° / pi mutant HB-EGF expressed in ° mice in experimental cell expression system such as previously, it has been confirmed that not liberated from the cell membrane without being degraded by protease. That is, HB-EGF pr . In the / p "mouse, the expressed HB-EGF remains in the cell membrane and is not released.
  • this HB_EG r . / pr By using mice, it is possible to analyze what abnormalities occur in the ecology of mice when HB-EGF is not released from the cell membrane and the EGF receptor cannot be stimulated. Therefore, the importance of the release of HB-EGF from the cell membrane can be examined.
  • mice that completely lacked HB-EGF function In this study, the HB-EGF genomic gene sequence was deleted, and the HB-EGF gene was inserted between the Krelli combinase recognition sequences using homologous recombination at the same site. As a result, a mouse having completely impaired HB-EGF gene function could be produced, and this mouse is hereinafter referred to as HB-EGF del / del mouse. Therefore, in one embodiment of the present invention, by using the HB-EGF del / del mouse, the action of HB-EGF in vivo can be further clarified.
  • HB-EGF pr ° / pr ° mice and HB-EGF del / del mice had marked heart failure
  • the pathological function of HB-EGF can be examined. This will clarify the role of HB-EGF in maintaining cardiomyocyte homeostasis and examine the importance of HB-EGF in the development of heart failure due to human dilated cardiomyopathy.
  • an epidermal growth factor (EGF) family factor such as HB-EGF and a substance that promotes its release from the cell membrane as a therapeutic agent for heart failure will be examined. Disclosure of the invention
  • the gist of the present invention is as follows.
  • a non-human animal having a DNA sequence of a heparin-binding epidermal growth factor-like growth factor gene into which a mutation that alters the function of the heparin-binding epidermal growth factor-like growth factor gene has been introduced.
  • the animal according to (6), wherein the heart failure is dilated cardiomyopathy characterized by expansion of the heart lumen and progression of cardiomyocyte degeneration and fibrosis.
  • cardiomyocyte degeneration occurs due to causes other than ischemia, hypertension, infection or autoimmune reaction due to vascular injury.
  • a cardiomyopathy model animal comprising the animal according to any one of (1) to (8).
  • a method for screening for a prophylactic and / or therapeutic agent for heart failure which comprises administering a test substance to the animal according to any one of (1) to (9).
  • a method for producing an animal comprising the following steps: DNA sequence of a heparin-binding epidermal growth factor-like growth factor gene into which a mutation that alters the function of the heparin-binding epidermal growth factor-like growth factor gene has been introduced.
  • heparin-binding epidermal growth factor-like growth factor into which a mutation that alters the function of the heparin-binding epidermal growth factor-like growth factor gene has been introduced.
  • the above method comprising producing a non-human animal having the DNA sequence of the gene.
  • the targeting vector has a DNA sequence of a part or the whole of the genomic DNA region of the heparin-binding epidermal growth factor-like growth factor gene sandwiched between clerical combinase recognition sequences.
  • a non-human cell having the DNA sequence of part or all of the genomic DNA region of the heparin-binding epidermal growth factor-like growth factor gene, flanked by reli compinase recognition sequences.
  • the non-human animal expressing crery combinase and the non-human animal are bred to obtain a part of the genomic DNA region of the heparin-binding epidermal growth factor-like growth factor gene or (12)
  • the targeting vector contains a cDNA for a heparin-binding epidermal growth factor-like growth factor into which a mutation that modifies at least one amino acid of the heparin-binding epidermal growth factor-like growth factor protein has been introduced. (12) The method according to any one of (16) to (16).
  • the targeting vector comprises a cDNA of a heparin-binding epidermal growth factor-like growth factor sandwiched between recognition sequences for Krelli combinase.
  • a prophylactic and / or therapeutic agent for heart failure comprising at least one of the following substances i) to Lii):
  • (22) Heparin-binding growth factor belonging to the family of epidermal growth factors
  • the animal of the present invention can be prepared by a gene targeting method.
  • Gene targeting is a method in which a part of the genomic DNA of the target HB-EGF gene is replaced with a separate gene DNA, and the original function of the gene on the genome is modified or deleted. . Therefore, in order to perform gene targeting, it is necessary to first isolate genomic DNA for the HB-EGF gene whose function is to be lost.
  • analysis of these animals preferably mice revealed that HB-EGF is essential for maintaining myocardial homeostasis, and conversely protects HB-EGF in areas where cardiac function has decreased due to myocardial damage. The possibility of working was suggested.
  • Genomic DNA is used to construct a targeting vector for homologous recombination of embryonic stem cells (ES cells). Therefore, it is desirable to isolate and use genomic DNA from the same animal species as the animal from which the ES cells are to be produced, so that recombination occurs more efficiently when performing homologous recombination. More desirably, in order to further increase the efficiency of homologous recombination, genomic DNA is isolated and used from animals of the same strain among animals of the same species from which ES cells are derived. This is because, even if the strains are the same, if the strains are different, DNA sequence mutations are scattered throughout the genome, and these mutations may reduce the probability of homologous recombination.
  • ES cells embryonic stem cells
  • a native cDNA or genomic DNA probe can be used to screen from a mouse genomic library.
  • a cDNA probe the full-length cDNA for the target HB-EGF gene or a part thereof may be used.
  • HB-EGF gene genomic band A bound to the probe with restriction enzymes and insert it into a commercially available cloning vector.
  • a cloning vector any of commercially available plasmids such as pBluescript, pBR322, and pUC may be used.
  • the targeting vector is used for homologous recombination of HB-EGF gene genomic DNA in ES cells with the modified HB-EGF gene. Therefore, the targeting vector for the HB-EGF gene in the present invention is required to contain a partially modified HB-EGF gene genomic DNA. Plasmid into which the genomic DNA of the HB-EGF gene has been introduced can be modified in vitro by modifying the genomic DNA sequence of the HB-EGF gene to remove the genomic DNA without performing steps such as ligation. can do.
  • modifying a part of the genomic DNA of the HB-EGF gene refers to causing a change, deletion, insertion or substitution in a part of the genomic DNA of the HB-EGF gene.
  • two or more methods of alteration, deletion, insertion or substitution of the HB-EGF gene may be used simultaneously.
  • alteration of genomic DNA refers to the genome of HB-EGF gene.
  • HB-EGF gene expression product has modified function as HB-EGF molecule That means.
  • ⁇ deletion '' of genomic DNA means that the expression product of the HB-EGF gene does not function or does not exist as an HB-EGF molecule by deleting a part or all of the genome of the HB-EGF gene. To do so.
  • “insertion” into genomic DNA refers to an HB-EGF gene in which a DNA having a sequence other than the HB-EGF gene is inserted into the HB-EGF gene to insert the DNA other than the HB-EGF gene.
  • -It means that the expression product of the EGF gene changes its function as an HB-EGF molecule.
  • substitution of genomic DNA means that part or all of the genome of the HB-EGF gene is replaced by a separate sequence that is not related to the HB-EGF gene, and the expression product of the HB-EGF gene is HB-EGF means that it does not function or is not present as a molecule.
  • screening of homologous recombinants includes (1) first-stage screening at the cell level in culture using a drug resistance gene introduced into a recombinant by a targeting vector, and (2) first screening. It is preferable that the recombinant selected by the single-stage screening is carried out in two steps of the second-stage screening at the DNA level, which is capable of performing PCR, Southern blot hybridization, and the like. Therefore, it is preferable to design the targeting vector so that the two-step screening can be performed more easily.
  • a targeting vector is produced by a method in which the genomic DNA sequence of the HB-EGF gene is replaced with a selectable marker gene DNA or the like. This is because a homologous recombinant obtained by homologous recombination with the DNA in the targeting vector can be more easily screened by using a selection marker or the like. More specifically, a portion of the genomic DNA of the isolated HB-EGF gene was deleted by restriction enzyme treatment, and the HB-EGF DNA and the modified HB-EGF DNA were substituted for the deleted DNA region. A targeting vector for the HB-EGF gene is prepared by inserting a selectable marker gene.
  • the promoter is not inserted in the vector, but it can be inserted into a DNA that replaces the promoter having strong expression activity in ES cells to facilitate selection of recombinants.
  • a selectable marker gene As a selectable marker gene, a neomycin resistance gene, a hygromycin B phosphotransferase gene, a diphtheria toxin A gene fragment for negative selection, and a lacZ gene can be used according to the purpose.
  • Selectable marker genes include, for example, neomycin resistance gene cassette (neo cassette, provided as pKJ2), hygromycin B phosphotransferase gene cassette (hph cassette), diphtheria toxin A gene cassette with thymidine kinase promoter (pMClDT).
  • -A selection marker gene that is commercially available inserted into a plasmid, such as lAZ gene cassette (provided as pBSlacZ) or lacZ gene cassette (provided as pBSlacZ) may be used.
  • the targeting vector may be constructed using any of the selectable markers linked to these promoters. It is also possible to use an endogenous promoter without using a promoter. Homologous recombination by targeting vector
  • homologous recombination of the HB-EGF gene means that a modified HB-EGF gene having the same or similar nucleotide sequence as the HB-EGF gene is artificially recombined on the HB-EGF gene genome. It means to let them.
  • Frequency of homologous recombination of the target gene occurs is known to be about 10 8.
  • it is necessary to perform the recombination operation theoretically 10 8 or more cells, the number at least
  • ES cells embryonic stem cells
  • ES cells it is preferable to use in the currently established gene targeting method. At present, only ES cells derived from mice have been produced, but if cells similar to mouse ES cells are established in various animals in the future, genetically modified animals can be produced using similar methods.
  • Mouse-derived ES cells that can be used at present include TT2 cells, AB-1 cells, J-1 cells, R-1 cells, and E14.1 cells. Which of the ES cells to use to create the targeting animal can be determined arbitrarily according to the purpose and method of the experiment, such as the mouse strain from which the collected genomic DNA is derived and the method for selecting chimeric animals. it can.
  • a targeting vector with a modified function of the HB-EGF gene is introduced into ES cells. Then, the genomic DNA sequence of the desired HB-EGF gene in the ES cell is replaced by homologous recombination with the functionally modified HB-EGF gene DNA sequence in the targeting vector. Homologous recombination can be stochastically generated using the homology between the HB-EGF gene genomic DNA sequence and the sequence of the non-modified portion in the targeting vector.
  • an electroporation method As a method for introducing a targeting vector into ES cells, an electroporation method, a calcium phosphate method, a DEAE-dextran method, or the like can be used. It is preferable to use the electroporation method in consideration of efficiency, workability, and the like.
  • selective culture which is the first stage of screening, is performed using the selection marker introduced into the ES cells by homologous recombination.
  • a plurality of selectable marker genes can be used among the selectable markers described above.
  • the culture medium for culturing ES cells should be changed daily.
  • the culture medium contains the selectable marker gene.
  • neomycin G418, Invitorogen
  • Hyigguchimycin B Calbiochem
  • a second screening step can be performed to confirm whether the target HB-EGF gene has been targeted.
  • the second screening is a step to confirm whether the target HB-EGF gene has been targeted at the DNA level.
  • a method there are a PCR method, a southern blot hybridization method, and the like.Either of them may be used, or a combination of two or more methods may be used. Is also good.
  • genomic DNA is prepared from each of the pools of ES cell clones selected by the first-stage screening, and the genomic DNA is used as type I to carry out a Southern blot hybridization method.
  • the second stage screening of ES cells is performed. Isolate clones that have undergone recombination by screening using this method.
  • probes are designed will depend on the particular selection marker used in constructing the targeting vector.
  • the number of probes used for Southern blot hybridization analysis can be determined to more reliably select a homologous recombinant. Creating knockout animals
  • the recombinant ES cells obtained as a result of the homologous recombination are transplanted into the 8-cell stage or blastocysts.
  • Put this ES cell transplanted embryo into the uterus of a pseudopregnant foster parent A chimeric animal can be produced by transplanting and giving birth.
  • the genetically modified animal is preferably a mouse, but is not limited thereto, and can be produced from various animals that can use the technology for genetically modifying a fertilized egg or embryo.
  • a method for transplanting the ES cells into the embryo for example, a micromanipulation method, an aggregation method, and the like are known.
  • the method of transplantation can be appropriately modified.
  • mice first, female mice superovulated with a hormonal agent (for example, using PMSG having FSH-like action and hCG having LH action) are mated with female mice. Then, collect embryonic embryos from the uterus 2.5 days after fertilization when using 8-cell stage embryos and 3.5 days after fertilization when using blastocysts. The embryos thus collected are injected in vitro with ES cells that have undergone homologous recombination using a targeting vector to produce chimeric embryos.
  • a hormonal agent for example, using PMSG having FSH-like action and hCG having LH action
  • a pseudopregnant female mouse to be a foster parent can be obtained by mating a normal-period female mouse with a male mouse castrated by vasectomy or the like.
  • a chimera animal can be prepared by implanting the chimera embryo prepared by the above-mentioned method into the uterus of the pseudopregnant mouse thus produced and giving birth to a pregnant and childbirth.
  • the female mouse that collects the fertilized egg and the pseudopregnant mouse that becomes the foster mother should be created from a group of female mice in the same estrous cycle. Desirable.
  • mice From such chimeric mice, select OS mouse derived from ES cell-transplanted embryos. After the selected chimeric mouse derived from the ES cell-transplanted embryo matures, the mouse is bred to a female mouse of a pure mouse strain, and the coat color derived from the ES cell appears in the next generation of offspring. Can be confirmed to have been introduced into the germline of Kimrungmas. Various traits can be used as indicators to confirm that ES cells have been introduced into the breeding line. Desirable.
  • mice wild boar (agouti), black (b lack), ocher (cinnamon), chocolate (choco late), and white (albino, albi no)
  • the mouse strain to be crossed with the chimeric mouse can be appropriately selected in consideration of the ES cell origin used.
  • a chimeric mouse of os created using ES cells derived from an agouti line (C57BL6 + CBA mouse) is mated with a black C57BL6 female mouse.
  • the coat color of the offspring of the next-generation offspring is wild gray (agouti) and black (black), which confirms that the ES cells have been introduced into the germ line of the chimeric mouse.
  • an animal in which a recombinant ES cell implanted in an embryo is introduced into a germ line is selected, and a chimeric animal is bred to obtain an individual deficient in a gene of interest.
  • a desired genetically modified homozygous mouse can be obtained.
  • mice in which the HB-EGF gene was replaced with a sequence containing the Krelli combinase recognition sequence were then transformed into mice that constitutively expressed Krelli combinase (Sakai K and Miyazaki J Biochem Biophys Res Commun. 237 : 318-24, 1997) to produce heterozygous mice deficient in the HB-EGF gene.
  • mice that constitutively expressed Krelli combinase Sakai K and Miyazaki J Biochem Biophys Res Commun. 237 : 318-24, 1997) to produce heterozygous mice deficient in the HB-EGF gene.
  • animals that have been established as strains that is, animals that are genetically homologous. Because the genetic background of such animals has been examined in detail, only the effects of treatment given to such animals under such known genetic backgrounds can be identified. On the other hand, it is difficult to determine whether the effect produced in a hybrid animal is due to the treatment alone or to the genetic background.
  • mice used for backcrossing can be appropriately selected by those skilled in the art. Examples of mice that can be used include, but are not limited to, BALB / c, C57BL6, DBA, and the like. In addition, it may take a long time if backcrossing is performed only by natural crossing, so in vitro fertilization technology can be used as appropriate when it is desired to accelerate generational replacement.
  • mice In each experiment using the produced genetically modified mice, it is preferable to use mature mice whose sex and age are matched. This is to eliminate the effects of gender and age-dependent factors on experimental results. In addition, experiments should be performed based on in-house ethical guidelines for animal experiments and safety guidelines for genetic engineering experiments.
  • the HB-EGF gene-modified mouse created as described above can be used in a test for examining the function of HB-EGF in the pathology of heart failure.
  • the HB-EGF gene-modified animal that develops heart failure of the present invention can be used not only for studying the development of heart failure, for example, dilated cardiomyopathy, but also for searching for a therapeutic agent for those diseases.
  • the present invention also provides a method of searching for a prophylactic and / or therapeutic agent for heart failure using the disease model animal of the present invention.
  • the search method of the present invention can be appropriately determined by those skilled in the art, and includes, for example, the following steps: a heart failure model animal, preferably a heart failure model mouse about 4 to 8 weeks old, a compound of a drug candidate or The substance can be administered intravenously, subcutaneously, intramuscularly, orally, etc. One or more doses per day, divided into one or several doses.
  • the compound or substance to be administered may be used by diluting it with a pharmaceutical solvent or a pharmaceutical diluting medium, or may be given as a mixture with water or feed.
  • Prevention or treatment of heart failure is judged based on suppression of disease onset or reduction of symptoms compared to model animals to which the same amount of vehicle or vehicle was administered in place of the candidate compound I do.
  • the test may include groups receiving a commercial treatment for heart failure such as angiotensin converting enzyme inhibitor or catecholamine beta receptor blocker as a positive control.
  • the present invention provides a prophylactic and / or therapeutic agent for heart failure, comprising at least one of the following substances i) to iii):
  • HB-EGF HB-EGF
  • EGF epidermal growth factor
  • amphiregulin ⁇ TGF-a transforming growth factor-
  • betacellulin epiregulin HB-EGF
  • neuregu ⁇ inl-6 etc.
  • HB-EGF EGF, araphiregulin ⁇ TGF- % betacellulin, and epiregulin neuregulinl-6 can be synthesized using an insect cell protein synthesis system. Specifically, a sequence containing all of the protein translation sites of these cDNAs was incorporated into a virus vector using Invitrogen's BAC-TO-BACTM Baculovirus Expression System, and then introduced into insect cells. Purify the growth factors in the culture supernatant. The synthetic protein may be synthesized using Escherichia coli, yeast, or vertebrate cells. Purification is performed by desalting the culture supernatant and then using a combination of ion exchange resin and reverse phase chromatography. Alternatively, it may be prepared by peptide synthesis. (Heo et al Protein Expr Purif. 2002
  • Meta-oral proteases can be obtained by synthesizing recombinant proteins from insect cells or Escherichia coli and purifying and adjusting them using a metal chelate column or an affinity column for inhibitors of meta-oral proteases (Fu et al Protein Expr Purif 200 U 21 (2): 268-74) 0
  • the substance of iii) is used as a therapeutic or prophylactic agent for heart failure, at least 90%, preferably 95% or more, more preferably 98% or more, and even more preferably It is preferable to use one purified to 99% or more.
  • the substances i) to iii) above are sterile with tablets, capsules, elixirs, mic orally capsules, orally, or with water or other pharmaceutically acceptable liquids. It can be administered parenterally in the form of injections, such as solutions or suspensions.
  • the substances i) to iii) can be formulated together with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like.
  • additives examples include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • Aqueous liquids for injection include, for example, saline, pudose and others.
  • Isotonic solutions containing adjuvants such as D-sorbitol, D-mannitol, sodium chloride, etc.
  • Suitable dissolution aids such as alcohol (eg, ethanol, etc.), polyalcohols (Eg, propylene glycol, polyethylene glycol, etc.) and non-ionic surfactants (eg, polysorbate 80 TM, HCO-50, etc.).
  • alcohol eg, ethanol, etc.
  • polyalcohols Eg, propylene glycol, polyethylene glycol, etc.
  • non-ionic surfactants eg, polysorbate 80 TM, HCO-50, etc.
  • oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a dissolution aid.
  • buffering agents eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalco-pum chloride, proactive hydrochloride, etc.
  • stabilizers eg, human serum albumin
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way may be human or warm-blooded animals (e.g., rats, mice, guinea pigs, puppies, birds, higgins, stags, puppies, pumas, cats, dogs, monkeys, chimpanzees, etc.). ) Can be administered.
  • the dosage varies depending on the target disease, the administration target, the administration route, and the like.
  • HB-EGF when HB-EGF is orally administered for the purpose of treating heart failure, it is generally used in adults (as 60 kg). It is recommended that HB-EGF be administered about 10 g-2 mg, preferably about 100 g-lmg per day, more preferably about 300 ⁇ g-500 / ig per day.
  • the single dose of HB-EGF varies depending on the subject of administration, the target disease, etc.
  • HB-EGF is administered in the form of an injection to an adult (body weight 6).
  • 0 g) of HB-EGF per day at about 1 / z g-200 / 2 g, preferably about 10 ⁇ g-100; xg, more preferably about 30 ⁇ g-50 ⁇ g It may be administered by injecting about g.
  • the amount can be administered in terms of the amount of the animal per body weight based on the dose for the above-mentioned adult (body weight of 6 O kg).
  • the present invention also relates to a vector into which a heparin-binding epidermal growth factor-like growth factor gene or a mutant thereof has been incorporated, A gene therapy agent for heart failure, comprising the vector capable of expressing a skin growth factor-like growth factor gene or a mutant thereof in an animal.
  • Heparin-binding epidermal growth factor-like growth factor gene can be prepared by a known method.
  • mouse heparin-binding epidermal growth factor-like growth factor gene can be prepared by the method described in Test Example 1 described below. , Can be clawed.
  • the DNA may be synthesized by a commercially available DNA synthesizer based on the DNA sequence information.
  • Mutants of the heparin-binding epidermal growth factor-like growth factor gene are those in which the nucleotide sequence of the heparin-binding epidermal growth factor-like growth factor gene is mutated, specifically, the addition of at least one base. DNA that is defective, defective, or substituted with another base.
  • This variant should encode a substance that has a biochemical effect similar to that of HB-EGF (eg, activates epidermal growth factor receptor).
  • Such a mutant can be obtained, for example, by deleting a part or the whole of a DNA sequence or inserting or substituting another DNA by a known genetic engineering technique.
  • the HB-EGF gene or a mutant thereof can be used as a drug for treating or preventing heart failure.
  • a patient in whom HB-EGF is reduced-deficient in a living body, or in a patient who is not supplied with a required amount of HB-EGF (1) administering the HB-EGF gene or a mutant thereof to the patient;
  • administering the HB-EGF gene or a mutant thereof to the patient;
  • By expressing HB-EGF in vivo (2) inserting the HB-EGF gene or a mutant thereof into cells, expressing HB-EGF, and then transplanting the cells into the patient, Role of HB-EGF can be fully or normally exerted.
  • the HB-EGF gene or its variant When using the HB-EGF gene or its variant as a therapeutic or prophylactic agent for heart failure, use the HB-EGF gene or its variant alone or in a retrovirus vector, adenovirus vector, or adenovirus associate. After insertion into a suitable vector such as a viral vector, it can be administered to humans or warm-blooded animals according to conventional means.
  • a suitable vector such as a viral vector
  • the HB-EGF gene or its variant can be formulated as is or with physiologically acceptable carriers such as supplements to promote uptake It can be administered via a catheter such as a gene gun or hydrogel catheter.
  • HB EGF genetically modified mice showing the construction of a fabrication targeting Tsu computing solid terpolymer.
  • FIG. 2 shows the results of a Southern blotting search of ES cells transfected with vectors for producing HB-EGF gene-modified mice (HB-EGF pr ° / pr . Mice). It can be seen that one of the homologous genes of the ES cells has been homologously recombined with the modified gene.
  • HB EGF genetically modified mice (HB- EGF pr. / Pr. Mice) base click coater for the production is made using ES cells introduced HB EGF genetic modification heterozygotes mice ( HB-EGF pr ° / + ), and their crosses
  • HB-EGF gene-modified homozygous mice (HB-EGF pWpf °) were collected from the tail
  • Figure 4 shows wild-type mice (HB-EGF + / + ) and HB-EGF gene-modified homozygotes.
  • Body mice HB- EGF pr. / Pr.
  • the expression of HB-EGF ⁇ Pi mutations HB EGF in each organ showing the result of investigation by Northern blotting.
  • HB-EGF pr . / pr ° expresses messenger RNA of mutant HB-EGF larger in size than HB-EGF + / + by gene modification.
  • HB-EGF 1 "expressed almost the same amount of mutant HB-EGF as HB-EGF + / + wild-type HB-EGF.
  • Figure 5 shows HB_EGF pr ° / pir .
  • 4 shows a comparison of survival curves for HB-EGF + / + and HB-EGF + / + .
  • FIG. 6 shows HB-EGF pi at 12 weeks of age.
  • the following shows the findings of HB-EGF + / + during thoracotomy.
  • HB-EGF pWpjr ° the heart is significantly enlarged compared to HB-EGF + / + .
  • FIG. 7 shows the heart of FIG. 6 fixed by perfusion with formalin and then removed.
  • HB-EGF ° / pr As for HB-EGF + / + , enlargement of the heart and atrial appendage was observed as compared with HB-EGF + / + .
  • Figure 8 shows HB_EGF at 12 weeks of age. 2 shows the physiological test results of HB_EGF + / + . HB-EGF P / Pf .
  • HB_EGF + / + the left ventricular lumen was enlarged and the contractility was decreased.
  • LVDd left ventricular end diastolic diameter, LVDs; left ventricular end systolic diameter, FS; left ventricular ejection fraction, sBP; systolic blood pressure, dBP; diastolic blood pressure, HR; heart rate
  • FIG. 9 shows the heart of FIG. 7 after paraffin embedding and sectioned at the level of the left ventricular papillary muscle and stained with hematoxylin-geosin.
  • HB-EGF pro / pro showed a larger left ventricular and right ventricular lumen than HB-EGF + / +, and the left ventricular wall was relatively thinner than the lumen.
  • FIG. 10 shows the same section as in FIG. 9 stained with Azanmalory and further enlarged. The site of fibrosis that stains blue is highlighted. HB-EGF pr . / pr . It is observed that fibrosis progresses transmurally in comparison with HB-EGF + / + .
  • FIG. 11 shows an enlarged image of the same section as in FIG. HB-EGF PJ: ° / Pr .
  • HB_EGF + / + fibrosis is remarkable and myocardial fibers are degenerated, resulting in large and small differences. No inflammation cell infiltration is observed.
  • FIG. 12 shows a targeting vector used for producing an HB-EGF gene-deficient mouse (HB-EGF del / del ).
  • K KpnI, V; EcoRV, NTR-lacZ; nuclear transporting peptide and internal ribosoma ⁇ enhance sequence-lacZ ⁇ PGK; phosph.0 glycerine kinase promoter
  • FIG. 13 shows the results of a Southern blotting search of ES cells into which the vector for producing the HB-EGF gene-deficient mouse (HB- EGFlQx / 1 ° x ) was introduced. It can be seen that one of the homologous genes of the ES cells was homologously recombined by the HB- EGFlQX allele.
  • Figure 14 shows the results of Southern blotting of DNA collected from the tail of various mice produced using ES cells transfected with vectors for HB-EGF gene-deficient mice (HB-EGF del del mice). The result of screening is shown.
  • HB-EGF 10 " 1 ⁇ ) was obtained by crossing mice heterozygous for HB-allele heterozygous (HB-EGF” 1 ") with HB-EGF 10 " 1 ⁇ ) mice, and HB-EGF " 1 ".
  • HB-EGF 10 " 1 ⁇ was obtained by crossing mice heterozygous for HB-allele heterozygous (HB-EGF” 1 ") with HB-EGF 10 " 1 ⁇ ) mice, and HB-EGF " 1 ".
  • HB_EGF del / del mouse Shows that a mouse completely deficient in the EGF gene (HB_EGF del / del mouse) could be produced.
  • Figure 15 shows the expression of HB-EGF in each organ of wild-type mouse (HB-EGF + / + ) and HB-EGF gene-deficient homozygous mouse (HB-EGF del del ) by RT-PCR. The results of the study are shown below. RT-PCR analysis of tissues revealed that HB-EGF del del did not substantially express HB-EGF messenger RNA due to gene deletion.
  • FIG. 16 shows the results of echocardiography of HB-EGF del / del and HB-EGF + / + at 12 weeks of age.
  • HB-EGF del / del had a larger lumen and reduced wall motion compared to HB_EGF + / + .
  • S represents systolic left ventricle diameter
  • d represents diastolic left ventricle diameter
  • FIG. 17 shows the hearts of HB-EGF del / del and HB-EGF + / + at 12 weeks of age after perfusion fixed with formalin and then removed.
  • HB-EGF del / del With HB-EGF del / del , enlargement of the heart and atrial appendage was observed as compared with HB-EGF + / + .
  • Figure 18 shows the degree of MTS reduction in rat cultured cardiomyocytes measured by absorbance. Higher absorbance indicates higher cell viability. 2 shows that the addition of HB-EGF increases the viability of cardiomyocytes.
  • mice are: (* P ⁇ 0.0001 vs H 2 0 2 + HBEGF -, #P rather O. OOl vs H 2 0 2 + HBEGF -, $ P ⁇ 0.05 vs H 2 0 2 + HBEGF-)
  • Example 1 A mouse in which HB-EGF is not released from the cell membrane in vivo by introducing a mutation into the target sequence of the protease when HB-EGF is released from the cell membrane (HB-EGF pr / pr .
  • Genomic DNA Cloning of HB-EGF Gene Genomic DNA for mouse HB-EGF was obtained from Clontech using the full-length mouse HB-EGF cDNA (SEQ ID NO: 5) as a probe. Isolated from a genomic phage library. Approximately 9 ⁇ 10 5 phage clones were screened by plaque hybridization and 30 positive clones were obtained. To determine whether these clones encoded HB-EGF, a second screening with the HB-EGF probe was performed separately.
  • HB-EGF positive clones derived from a single phage clone were obtained.
  • a genomic clone structural map was prepared by digestion of the resulting clone with some restriction enzymes and partial sequencing (Fig. 1).
  • the resulting HB-EGF clone covered 30.4 kb of the gene and contained homologous sequences corresponding to exons 1 to 6 of the human HB-EGF gene.
  • FIG. 1 shows the targeting strategy of the HB-EGF gene.
  • HB- EGF To create a targeting vector for the gene, a portion of the isolated genomic clone was deleted with a restriction enzyme and the 148th lysine of the HB-EGF protein was added to the serine. 4 HB-EGF cDNA ( ⁇ HB-EGF) (SEQ ID NO: 6) into which the ninth proline is changed to threonine, and a positive selection marker gene (IRES-neopolyA cassette) (IRES Is an abbreviation of International ribosome entry site-SEQ ID NO: 7, polyA means poly A).
  • Is an abbreviation of International ribosome entry site-SEQ ID NO: 7 means poly A.
  • the construction of the targeting beta of HB-EGF consists of the steps from the N-terminal coding region for mature HB-EGF to Ethason 3 between the SacI I and HindIII sites in etason 1 and intron 3, respectively.
  • the DNA fragment of about 6 kb was deleted, and a ⁇ HB-EGF-IRES-neo-polyA cassette was inserted in place of the DNA fragment.
  • a diphtheria toxin A fragment gene (DT) was inserted into the 3 'end of the vector under the control of the thymidine kinase promoter as a negative marker (Fig. 1).
  • the homology regions at the 5 'and 3' ends were about 7 kb and 4 kb, respectively.
  • ES cells used in the present invention were obtained from C57BL6 + CBA mice.
  • TT2 cells can be obtained as described in Dr. Shinichi Aizawa (Experimental Medicine Separate Volume Biomanual Series 8, Generation of Mutant Mice Using Gene Targeting Guo ES Cells, Yodosha, 1995).
  • neomycin 200 At g / ml, Sigma
  • Dulbecco's modified Eagle medium Nacalai Tester
  • thamelcaptoethanol Nacalai Tesque
  • the medium containing the drug is changed daily.
  • 116 ES clones grown in the presence of neomycin were pooled and genomic DNA was isolated from them. To confirm whether homologous recombination had occurred, Kakuin screened using the Southern blot hybridization method.
  • the fragment amplified with the primers (5'-GAGACCCCCATCTCTATGACAG-3 '(SEQ ID NO: 9) and 5, -TCCGGAAGCTCTGTCTTTCAC-3' (SEQ ID NO: 10)) was used as the 5 'probe (probe A in Fig. 1).
  • the PvuI I-PvuI I-fragment as a 3 'probe (probe B in Fig. 1)
  • Southern blot hybridization analysis was performed on the targeted HB-EGF gene.
  • Genomic MA obtained from ES cells is digested with Hindlll, electrophoresed on a 0.5% agarose gel, and then transcribed to Nia Membrane (Amershara).
  • Hybridization was performed using probes A and B labeled with 32 P in hybridization buffer (Toyobo) 68. Perform in C. After washing, the membrane was exposed to a film and detected.
  • hybridization buffer Toyobo
  • the membrane was exposed to a film and detected.
  • the expected DNA bands of probe A) and lkb (probe B) were detected (Fig. 2). This indicated that one of the wild-type alleles in the targeted ES clone had been correctly replaced by the mutant gene.
  • the targeting efficiency of the HB-EGF gene was 6.0%. Of these, the introduction of the targeted gene into the germ line was confirmed in two out of two clones.
  • ES cells targeting the HB-EGF gene are transplanted into fertilized embryos.
  • the method of transplantation can be appropriately modified by those skilled in the art. However, as described in the present Example, the report of Origininale (Nagy et al., 1993, Proc. Acad. ScI. USA 90, 8424-8428), a chimeric embryo was produced by the injection method for an 8-cell stage embryo.
  • the lineage of the embryo that can be used as the 8-cell stage embryo in the injection method may be a line derived from a cross between pure lines or a line derived from a cross between pure lines. In the present invention, embryos derived from F1 of ICR were used.
  • the mouse After maturation of the OS chimeric mouse derived from the ES cell-transferred embryo, the mouse is mated with a female mouse of a pure mouse strain, and ES cells are introduced into the germline of the chimeric mouse by the coat color of the next generation of offspring. I confirmed that.
  • the created chimeric mouse of oss was bred with black C57BL6 mouse. Then, it was confirmed that the ES cells were introduced into the germ line of the chimeric mouse by the color of the coat color of the offspring of the next generation offspring (agouti) + black (black).
  • TT2 agouti line
  • HB_EGF pr homologously recombined in mice obtained by crossing heterozygotes. Screening was performed using PCR using thigh A isolated from the tail.
  • the PCR reaction was performed using primers P1: 5'-AGGGCAAGATCATGTGTCCTGCCTCAAGCC-3 '(SEQ ID NO: 11) and P2: 5'-GCCCCTCCCCCGTGCCTTCCTTGAC-3' (SEQ ID NO: 1).
  • PCR reaction buffer was 50 mM Tris-HC1 (pH 8.0), 0.1 mM EDTA, lmM DTT, 0.001% Tween 20, 0.001% NP—40, 50% , Riseronore, was carried out at 25mMMgS0 4, 2raMdNTP and 1 U / / 1 of the polymerase (KODP lu s, Toyobo).
  • PCR cycle conditions were as follows: 30 cycles at 94 ° C for 15 seconds, 62 ° C for 30 seconds, and 68 ° C for 1 minute as one cycle. As shown in FIG. 3, HB-EGF pr was found in the heteroconjugate.
  • homozygotes had both the HB-EGF pro allele and the wild-type allele only.
  • homozygotes HB-EGF pr ° / pr . Mice
  • HB-EGF + / + mice wild-type mice
  • HB-EGF pr ° / pi ". Mice mice
  • Northern blotting of each tissue was determined by Northern blotting of each tissue.
  • Menpuren is 2 x SSC, 0 After washing twice with 1% SDS solution (60 ° C) for 5 minutes, the film was exposed to light and detected.
  • the expression level of the modified gene and the expression pattern in each organ may change due to the introduction of the foreign gene, in this example, as shown in FIG.
  • mutant HB-EGF was expressed in each organ almost as much as in HB-EGF + / + mice.
  • the expression of the mutant HB-EGF mRNA was larger than that of the non-mutated HB-EGF, so that the respective expressions could be clearly distinguished.
  • HB-EGF. / In mice, the lower band was barely detectable, indicating that the expression of unmutated HB-EGF was almost completely inhibited. That is, HB-EGF pr . / pr . In mice, the expression of HB-EGF without mutation was almost completely suppressed by gene modification, confirming that only mutant HB-EGF not released from the cell membrane was expressed. It was also shown that the expression level was comparable to HB-EGF without HB-EGF + / + mouse mutation in each organ. It was confirmed that HB-EGF mutant homozygous mice (HB-EGF pr ° / pr . Mice) did not seriously impair fetal development, but died early. The survival curves were compared with wild-type mice (HB-EGF + / + mice) bred under the same conditions (Fig. 5). HB-EGF pr . / pr . Half of the mice died at about 18 weeks.
  • HB-EGF pWpir ° mice have a diminished motor capacity a few days before death, and female HB-EGF pr ° / p mice have a cardiac load that may be due to perfusion of the uterine bloodstream early after delivery.
  • HB-EGF P1 "° / Pr often indicates death from heart failure. This suggests weakness in the heart function of mice.
  • HB_EGF pr by echocardiographic observation. / pr .
  • the left ventricular lumen was significantly dilated during both systolic and diastolic phases, and wall motion was decreased to all rounds compared to HB-EGF + / + mice.
  • the blood pressure tended to decrease slightly, and the heart rate tended to increase slightly, which was considered to reflect a decrease in cardiac function.
  • mice were perfusion-fixed with 4% paraformaldehyde, then infiltrated and fixed for 1 mm, dehydrated, and embedded in paraffin. Sections of 6 ⁇ m were prepared by microtome and observed by Hematoxylin 'eosin staining and Azanmalori monostaining.
  • HB-EGF pr ° / p mice 12 weeks after birth, were fixed by refluxing with 2.5% glutaraldehyde, embedded in ebon resin, and sliced at about 30 nm with an ultramicrotome.
  • Non-specific findings such as loss of contractile structure and changes in mitochondria were found in myocardium degenerated by electron microscopy, but findings suggestive of storage disease, interstitial abnormalities such as amiloidosis, etc. was not observed.
  • the HB_EGF pro / pro mouse is a model animal very similar to human idiopathic dilated cardiomyopathy for the following reasons. First of all, HB-EGF pr . / pr . Mice other conditions that cause c then myocardial degeneration secondarily early death by heart failure leading to degeneration of the heart muscle cells themselves onset in young, for example, hypertension, autoimmune myocarditis, infectious myocarditis, imaginary The involvement of blood cardiomyopathy, accumulation diseases such as amyloidosis and Fabry disease, etc. seems to be extremely low based on physiological and histological studies.
  • mice can be mainly used as a model animal for dilated cardiomyopathy, which mainly develops myocardial cell degeneration, for elucidating the pathology of heart failure and for screening drugs for heart failure.
  • Example 2 Creation of HB-EGF gene deficient mouse; Genomic DNA fragment of HB-EGF gene Construction of cloning and targeting vectors
  • the same genomic DNA as that in Test Example 1 was used for mouse HB-EGF.
  • the construction of the targeting vector for HB-EGF is approximately 6 kb, including the N-terminal coding region for mature HB-EGF between SacI I and EcoRV sites in Ethason 1 and Intron 3 to Ethason 3. ⁇ Remove the A fragment, and insert the loxP—HB—EGF—polyA—loxP—lacZ—pA cassette (HB_EGF cDNA region sandwiched between loxP and loxP sequences is shown as SEQ ID NO: 8).
  • the lacZ gene which is connected to the nuclear transporting peptide and internal ribosome enhance sequence, was inserted into the DNA fragment instead of the DNA fragment.
  • the neomycin gene under the PGK promoter was introduced on the 3 'side of the vector. Furthermore, a diphtheria toxin A fragment gene (DT) was inserted as a negative marker at the 5 'and 5' ends of the vector under the control of the thymidine kinase promoter (Fig. 12). The homology regions at the 5 'and 3' ends were about 7 kb and 9 kb, respectively.
  • the ES cells used in this example are TT2 cells obtained from C57BL6 + CBA mice. TT2 cells were obtained as described in Dr. Shinichi Aizawa (Experimental Medicine Separate Volume Biomanual Series 8, Gene Targeting-Generating Mutant Mouse Using ES Cells, Yodosha, 1995). By performing electroporation on ES cells, about 10 7 cells
  • HB-EGF gene-targeted ES cells into a fertilized embryo.
  • the transplantation method can be appropriately modified by those skilled in the art, in this example, the original report (Nagy et al., 1993, Proc. Acad. ScI. USA 90, 8424-8428), and chimeric embryos were prepared by the injection method for 8-cell stage embryos. That is, put ES cells in the hole on the plastic dish between the 8-cell stage transparent body that has not undergone compaction and the blastomere, and incubate in BBW medium or M16 medium, and then Well-formed blastocysts were implanted into the offspring of pseudopregnant female mice (Hogan et al.
  • the lineage of the embryo that can be used as the 8-cell stage embryo in the injection method may be a line derived from a cross between pure lines or a line derived from a cross between pure lines.
  • an embryo derived from F1 of ICR was used.
  • the mouse After maturation of the OS chimeric mouse derived from the ES cell-transferred embryo, the mouse is bred with a female mouse of a pure mouse strain, and ES cells are introduced into the germ line of the chimeric mouse by the coat color of the next-generation offspring. I confirmed that.
  • the strain derived from the wild gray (agouti) strain (TT2) was used.
  • HB-EGF del / de I Homozygous mice lacking the HB-EGF gene
  • the genomic DNA of the tail was collected from each individual, and it was confirmed by Southern blotting whether HB-EGF was correctly deleted.
  • Collected Ge Nom DNA was digested with Kpnl, digested with Kpnl, expanded by electrophoresis, and 3'primer (5'-ACCCACACTCCAGTCACGGCTGC-3 '(SEQ ID NO: 17) and 5, primer 5'-CGGTGGAGGACAGCGAGGTTCCAC-3' The fragment amplified in No. 18))) was used as a Lox probe and detected by Southern blotting.
  • HB-EGF del / del mice were also found to be born according to the Mendelian ratio in crosses of the heterozygote zygotes. This indicates that deficiency of HB-EGF does not cause significant abnormalities in embryonic development.
  • HB _ E GF dei / dei mouse is not critical failure is found in the fetal period occurs, it has been confirmed that the death at an early stage. Echocardiography at 12 weeks after birth showed dilated heart and decreased peripheral wall motion (Fig. 16).
  • HB_EGF pr . / pr Since mice and HB-EGF del / del mice exhibited similar phenotypes (heart failure due to cardiomyocyte degeneration), HB-EGF bound to the cell membrane was degraded by proteolytic enzymes to maintain cardiomyocyte function. Therefore, it was shown that it is necessary to become free form and act. So HB-EGF is the heart Muscle-specifically plays an important role in maintaining its function, suggesting that compounds that promote HB-EGF supplementation and HB-EGF release can be used for the treatment of heart failure.
  • rat cultured cardiomyocytes were examined.
  • the cells collected by centrifugation were redispersed in DMEM cells and incubated at 37 ° C in 95% air-5% carbon dioxide saturated steam for 1 hour. After removing the fibroblasts, the obtained cardiomyocytes were seeded in 96 wells.
  • the medium was replaced with a DMEM culture solution containing 10% calf serum, and then the cells were cultured for 3 days.
  • the medium was replaced with DMEM culture medium without serum (100 ⁇ 1 / wel l) on day 4, 10- 5 M re con Binanto HB EGF on day 5 of culture, 10- 6 ⁇ , 1 ( ⁇ 8 ⁇ , 10-1 () was added at a concentration of Micromax, were cultured for 18 hours.
  • HB-EGF had an excellent protective effect against myocardial damage due to the oxidative stress of hydrogen peroxide. Therefore, Substances that cause EGF receptor activity, such as HB-EGF, are useful in various heart diseases such as ischemic heart disease such as myocardial infarction, myocardial damage during ischemic conditions during cardiac surgery, and heart failure resulting from various diseases. Useful as a therapeutic, diagnostic and prophylactic agent.
  • HB-EGF not only causes serious myocardial damage due to its deficiency, but also has an excellent myocardial protective action against myocardial damage caused by oxygen radicals and the like. Therefore, the HB-EGF of the present invention is useful as an agent for treating or preventing various heart diseases that cause heart failure such as idiopathic dilated cardiomyopathy, ischemic heart disease such as myocardial infarction, myocarditis and the like. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability
  • the HB-EGF gene-modified mouse of the present invention can be used for a test for examining the function of HB-EGF in a pathological condition of heart failure.
  • the HB-EGF gene-modified animal that develops heart failure of the present invention can be used not only for studying the development of heart failure, for example, dilated cardiomyopathy, but also for searching for a therapeutic agent for those diseases. Sequence listing free text
  • SEQ ID NO: 1 shows the genomic sequence of the mouse HB-EGF gene including exons 1 and 2.
  • SEQ ID NO: 2 shows the genomic sequence of mouse HB-EGF gene containing exon 3.
  • SEQ ID NO: 3 shows the genomic sequence of mouse HB-EGF gene including exon 4.
  • SEQ ID NO: 4 is the genome of mouse HB-EGF gene containing exons 5 and 6. Shows the sequence.
  • SEQ ID NO: 5 shows the nucleotide sequence of cMA for mouse HB-EGF mRNA.
  • SEQ ID NO: 6 shows the nucleotide sequence of a mutant cDNA of mouse HB-EGFpro allele.
  • SEQ ID NO: 7 shows the IRES sequence.
  • SEQ ID NO: 8 shows the nucleotide sequence of a mutant cDNA of mouse HB-EGF lox out allele.
  • SEQ ID NO: 9 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 10 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 11 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 12 shows the nucleotide sequence of the primer.
  • SEQ ID NO: 13 shows the nucleotide sequence of lox P site.
  • SEQ ID NO: 14 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 15 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 16 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 7 shows the nucleotide sequence of a primer.
  • SEQ ID NO: 18 shows the nucleotide sequence of a primer.

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Abstract

L'invention concerne un animal non-humain possédant une séquence d'ADN d'un gène de facteur de croissance de type facteur de croissance épidermique se liant à l'héparine dans laquelle une mutation de modification de la fonction du gène de facteur de croissance de type facteur de croissance épidermique se liant à l'héparine a été transférée et un procédé de production d'un tel animal. L'invention concerne également des cellules souches embryonnaires non-humaines possédant une séquence d'ADN d'un gène de facteur de croissance de type facteur de croissance épidermique se liant à l'héparine dans laquelle une mutation de modification de la fonction de ce gène a été transférée, un procédé de criblage d'un agent préventif et/ou d'un remède contre l'insuffisance cardiaque, ainsi que cet agent et/ou ce remède.
PCT/JP2003/005672 2002-05-16 2003-05-07 Facteur de croissance d'animal genetiquement modifie de type facteur de croissance epidermique se liant a l'heparine, procede de criblage, cellules souches embryonnaires et agent preventif et/ou remede contre l'insuffisance cardiaque WO2003096804A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003235855A AU2003235855A1 (en) 2002-05-16 2003-05-07 Heparin-binding epidermal growth factor-like growth factor gene-modified animal, screening method, embryonic stem cells and preventive and/or remediy for heart failure
JP2004504814A JPWO2003096804A1 (ja) 2002-05-16 2003-05-07 ヘパリン結合性上皮増殖因子様増殖因子遺伝子機能改変動物、スクリーニング方法、胚性幹細胞、並びに心不全予防および／または治療薬

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JP2002-141791 2002-05-16
JP2002141791 2002-05-16

Publications (1)

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WO2003096804A1 true WO2003096804A1 (fr) 2003-11-27

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PCT/JP2003/005672 WO2003096804A1 (fr) 2002-05-16 2003-05-07 Facteur de croissance d'animal genetiquement modifie de type facteur de croissance epidermique se liant a l'heparine, procede de criblage, cellules souches embryonnaires et agent preventif et/ou remede contre l'insuffisance cardiaque

Country Status (3)

Country Link
JP (1) JPWO2003096804A1 (fr)
AU (1) AU2003235855A1 (fr)
WO (1) WO2003096804A1 (fr)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Soyakuto Human Science Kenkyu Juten Kenkyu Hokokusho, Heisei 13 Nendo, 2nd Field, Soyaku no Tameno Seitai Kino Kaiseki ni Kansuru Kenkyu", September 2002, article EISUKE MEKATA ET AL.: "Hen'igata HB-EGF knockin mice ni yoru junkanki shikkan no kaiseki", pages: 174 - 178, XP002972136 *
ABRAHAM J.A. ET AL.: "Heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones and transcript expression in tissues", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 190, no. 1, 1993, pages 125 - 133, XP002972138 *
CAPECCHI M.R.: "Altering the genome by homologous recombination", SCIENCE, vol. 244, no. 4910, 1989, pages 1288 - 1292, XP002972139 *
IWAMOTO R. ET AL.: "Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function", PROC. NATL. ACAD. SCI. USA, vol. 100, no. 6, 18 March 2003 (2003-03-18), pages 3221 - 3226, XP002972137 *
KURISAKI T. ET AL.: "Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis", MOL. CELL BIOL., vol. 23, no. 1, January 2003 (2003-01-01), pages 55 - 61, XP002960968 *
SUMIO KAWATA ET AL.: "Kimo setsujogo no zoshoku inshi network to atarasii kimo saisei inshi HB-EGF", RINSHO KAGAKU, vol. 34, no. 9, 1998, OSAKA, pages 1229 - 1235, XP002972140 *

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JPWO2003096804A1 (ja) 2005-09-15

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