JP2004166596A - Zaq gene-modified animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a model animal useful for screening a medicine for preventing/treating various kinds of diseases caused by deletion of ZAQ. <P>SOLUTION: The ZAQ gene expression-insufficient nonhuman mammal is the animal having the ZAQ gene inactivated by a reporter gene. The method for screening the medicine for preventing/treating the various kinds of the diseases caused by the deletion of the ZAQ uses the nonhuman mammal. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】野生型アレル（上図）、ＺＡＱターゲティングベクター（中図）および相同組換え体アレル（下図）の構造を示す概略図である。
黒のボックスはＺＡＱのコーディングエクソンを示す。白のボックスは外来遺伝子の発現カセットを示し、ボックス内の矢印は転写方向を示す。Ｎｅｏはネオマイシン耐性遺伝子、ＤＴＡはジフテリア外毒素遺伝子をそれぞれ示す。
【図２】選抜されたＥＳ細胞株がＺＡＱ遺伝子ヘテロ欠損相同組換え体であることを示すサザン解析結果を示す写真である。
ＥＳ細胞５株（Ｎｏ．１２２， １６３， ２７４， ２８７， ４１８）および野生株（Ｗｉｌｄ）よりそれぞれゲノムＤＮＡ を抽出してＢａｍ ＨＩで切断し、３’側プローブ（図１参照；左パネル）、および５’側プローブ（図１参照；右パネル）を用いてサザン解析を行った。前者では５株すべてに１０．６および６．５ ｋｂ ２本のバンドが認められ、後者では５株すべてに１０．６および ４．１ ｋｂの２本のバンドが認められたので、Ｎｏ．１２２， １６３， ２７４， ２８７および４１８の５個のＥＳ細胞株はＺＡＱ遺伝子ヘテロ欠損相同組換え体であることが明らかになった。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a non-human mammal embryonic stem cell in which the ZAQ gene is inactivated, a non-human mammal deficient in ZAQ gene expression, a screening method using the same, and a prophylactic / therapeutic agent obtainable by the screening.
[0002]
[Prior art]
Advances in genetic engineering technology and the rapid accumulation of knowledge of molecular biology as the basis thereof have made it possible to artificially manipulate genes and further introduce them into animal individuals (Non-Patent Document 1). For example, methods have been developed to artificially add exogenous genetic traits that are not originally present in the organism, or to suppress the expression of endogenous genetic traits possessed by the organism. Animals with various introduced genetic traits have been produced and reported as transgenic animals.
Such transgenic animals enable individual-level studies of gene functional analysis, which previously had limited knowledge obtained from in vitro cultured cells such as cell lines and primary cultured cells. Therefore, it is important in clarifying the functions of various genes that have been isolated and cloned by techniques such as genetic engineering. In particular, experiments and studies using this transgenic animal as an analysis of the physiological function of the cloned gene in an organism and a model system for a genetic disease have become active.
[0003]
Embryonic stem cells (hereinafter, abbreviated as ES cells) are established from an inner cell mass present inside a blastocyst, which is an early embryo after fertilization, and can be grown and cultured while maintaining an undifferentiated state. Cell line. These cells have pluripotency (totipotency) that can differentiate into all kinds of cells in the living body, and when injected into normal early embryos, they can participate in embryo body formation and form chimeric animals .
Utilizing this property, creation of various genetically modified animals has been attempted. The history began with the establishment of ES cells by Evans and Kaufman in 1981 (Non-patent document 2), and the study was started in earnest by the creation of ES chimeric mice by Bradley et al. (Non-patent document 3). Furthermore, the homologous recombination of ES cells by Thomas and Capecchi (Non-patent Document 4), the success of Germ line Transmission of ES cell traits by three research groups such as the group of Koller et al. (Non-patent Document 5), the generation of gene-deficient mice Research progressed rapidly.
ES cells that have been established to date include Evans and Kaufman EK cells (Non-patent Document 2), Doetschman ES-D3 cells (Non-patent Document 6), and Robertson CCE cells (Non-patent Document 6). Patent Literature 7), BL / 6III cells of Ledermann and Burki (Non-Patent Literature 8), and the like, most of which are established from 129 strain mice.
[0004]
Examples of such gene-deficient animals produced using ES cells include (1) HPRT gene deficiency using spontaneously mutated ES cells of Hooper et al. (Non-Patent Document 9) and Knehn et al. (Non-Patent Document 10). Mouse, (2) p53-deficient mouse lacking p53 which is one of the tumor suppressor genes of Donehower et al. (Non-patent Document 11), (3) β2 microglobulin gene mutant mouse of Zijlstra et al. (Non-patent Document 12) (4) RAG-2 (V (D) J recombination activation gene) mutant mouse of Sinkai et al. (Non-patent document 13), which is one of immune disease model mice, (5) Glimcher et al. (Non-patent document 14) and (6) MHC class II mutant mouse of Cosgrove et al. -As one of the development-related disease model mice, an int-1 gene-deficient mouse such as MacMahon et al. (Non-patent Document 16) and a src-deficient mouse exhibiting marble-like symptoms of (7) Soriano et al. Have been.
[0005]
Non-Patent Document 18 describes mouse-derived GPR73 (ZAQ1).
Patent Literature 1 describes mouse-derived mI5E (ZAQ2).
Patent Literature 2 describes rat-derived ZAQ1 and ZAQ2.
[0006]
[Patent Document 1]
International Publication No. 98/46620 pamphlet
[Patent Document 2]
WO 02/16607 pamphlet
[Non-patent document 1]
Gordon J. W. (Gordon, JW) et al., Proc. Natl. Acad. Sci. USA, United States, 1980, 77th. Vol., P. 7380-7384
[Non-patent document 2]
Evans M. J. (Evans MJ) and Kaufman M. H. (Kaufman MH), Nature, UK, 1981, vol. 292, p. 154-156
[Non-Patent Document 3]
Nature, UK, 1984, Volume 309, p. 255
[Non-patent document 4]
Cell, USA, 1987, vol. 51, p. 503
[Non-Patent Document 5]
Procedurals of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), USA, 1989, Vol. 86, p. 8927
[Non-Patent Document 6]
Journal of Embryology and Experimental Morphology (J. Embryol. Exp. Molfol.), UK, 1981, Vol. 87, p. 27
[Non-Patent Document 7]
Nature, UK, 1986, Volume 323, p. 445
[Non-Patent Document 8]
Experimental Cell Research, USA, 1991, Vol. 197, p. 254 pages
[Non-Patent Document 9]
Nature, UK, 1987, Vol. 326, p. 292
[Non-Patent Document 10]
Nature, UK, 1987, Vol. 326, p. 295
[Non-Patent Document 11]
Nature, UK, 1992, Volume 356, p. 215
[Non-Patent Document 12]
Nature, UK, 1990, Volume 344, p. 742
[Non-patent document 13]
Cell, USA, 1992, Vol. 68, p. 855
[Non-patent document 14]
Science, USA, 1991, vol. 253, p. 1417
[Non-Patent Document 15]
Cell, USA, 1991, Vol. 66, p. 1051
[Non-Patent Document 16]
Cell, USA, 1990, Vol. 62, p. 1073
[Non-Patent Document 17]
Cell, USA, 1991, Vol. 64, p. 693
[Non-Patent Document 18]
Biochimica Biophys. Acta, Netherlands, 2000, Vol. 1491, p. 369-375
[0007]
However, there are many points left to be elucidated further about the function and action mechanism of ZAQ in vivo. In order to elucidate the effects of ZAQ on the living body, a model of a ZAQ gene-deficient animal that does not produce ZAQ at all or produces only a small amount of ZAQ has been craved as an essential one. No animal model has been created.
[0008]
[Problems to be solved by the invention]
Therefore, if the non-human animal ES cells in which the ZAQ gene is inactivated are successfully prepared, a non-human animal lacking the expression of the ZAQ gene can be produced. Since the obtained non-human animal lacking ZAQ gene expression lacks various biological activities that can be induced by ZAQ, it serves as a model for diseases caused by inactivation of biological activities of ZAQ, Investigation and treatment methods can be studied.
[0009]
[Means for Solving the Problems]
In view of the above problems, the present inventors have made intensive studies and succeeded in producing a non-human animal deficient in ZAQ gene expression, and as a result of further studies, completed the present invention.
That is, the present invention
[1] a mammalian embryonic stem cell in which the ZAQ gene has been inactivated,
[2] the embryonic stem cell according to the above [1], which is drug-resistant;
[3] The embryonic stem cell according to the above [2], wherein the drug is neomycin;
[4] The embryonic stem cell according to [1], wherein the ZAQ gene is inactivated by insertion of a reporter gene,
[5] The embryonic stem cell according to the above [4], wherein the reporter gene is a lacZ gene,
[6] the embryonic stem cell according to the above [1], wherein the mammal is a mouse;
[7] The embryonic stem cell according to the above-mentioned [1], wherein the ZAQ gene is a gene containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
[8] a non-human mammal deficient in ZAQ gene expression,
[9] The animal of the above-mentioned [8], wherein the ZAQ gene is inactivated by insertion of a reporter gene,
[10] The animal of the above-mentioned [9], wherein the reporter gene is under the control of a promoter for the ZAQ gene,
[11] The animal according to the above [8], wherein the non-human mammal is a mouse,
[12] The animal of the above-mentioned [8], wherein the ZAQ gene is a gene containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
[13] Compared to the corresponding ZAQ gene-expressing animal,
(1) In a female animal, a fertilized egg cannot be stably obtained by superovulation, and / or
(2) When fertilized eggs are cultured, the incidence of early embryos is low.
The animal according to any one of [8] to [12], which has the phenotype of
[14] A substance for preventing or treating a gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising using the animal or the tissue thereof or the cell derived therefrom according to the above [8]. Screening method,
[15] The screening method according to [14], which comprises administering a test compound to the animal according to [8], a tissue thereof, or a cell derived therefrom,
[16] A substance for preventing or treating a digestive organ disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease obtained by the screening method according to the above [14] or [15],
[17] A prophylactic / therapeutic agent for a digestive organ disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising the prophylactic / therapeutic substance according to the above [16].
[18] A disease model animal produced by crossing the animal according to the above [8] with another disease model animal,
[19] A disease state model animal caused by drug induction or stress load on the animal according to the above [8],
[20] A pathological model animal caused by drug induction or stress load on the animal according to the above [18],
[21] A gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency, or a disease, characterized by using the animal according to any one of the above [18] to [20] or a tissue thereof or a cell derived therefrom. A method for screening a substance for preventing or treating a neurological disease,
[22] A gastrointestinal disease, an endocrine disease, a gonadal disease, or a cancer, which comprises administering a test compound to the animal or the tissue thereof according to any one of the above [18] to [20] or a cell derived therefrom. A method for screening a substance for preventing or treating an immunodeficiency or a neurological disorder,
[23] A prophylactic / therapeutic substance for a digestive organ disease, endocrine disease, gonadal disease, cancer, immunodeficiency or neurological disease obtained by the screening method according to the above [21] or [22],
[24] A prophylactic / therapeutic agent for a gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising the prophylactic / therapeutic substance according to the above [23].
[25] A method for screening a compound or a salt thereof that promotes or inhibits promoter activity for a ZAQ gene, comprising administering a test compound to the animal according to the above [10], and detecting expression of a reporter gene;
[26] A compound or a salt thereof that promotes or inhibits a promoter activity for a ZAQ gene obtainable by the screening method according to the above [25],
[27] A gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease comprising a compound or a salt thereof that promotes the promoter activity of the ZAQ gene obtainable by the screening method according to the above [25]. Prophylactic and therapeutic agents, and
[28] A gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease comprising a compound or a salt thereof that inhibits the promoter activity for the ZAQ gene obtainable by the screening method according to the above [25]. To provide prophylactic / therapeutic agents.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
[ZAQ gene expression inactive mammalian ES cells]
ZAQ includes ZAQ1, ZAQ2, and the like.
As the ZAQ1 gene, for example,
(1) a mouse ZAQ1 gene (genomic DNA) containing the nucleotide sequence represented by SEQ ID NO: 2, encoding mouse ZAQ1 containing the amino acid sequence represented by SEQ ID NO: 1;
(2) a rat ZAQ1 gene (cDNA) encoding the rat ZAQ1 containing the amino acid sequence represented by SEQ ID NO: 3 and containing the base sequence represented by SEQ ID NO: 4 (Patent Document 2);
(3) a mouse GPR73 (ZAQ1) gene (cDNA) containing the nucleotide sequence represented by SEQ ID NO: 5, encoding the mouse GPR73 (ZAQ1) containing the amino acid sequence represented by SEQ ID NO: 1 18) is used.
As the ZAQ2 gene, for example,
(1) a rat ZAQ2 gene (cDNA) containing the nucleotide sequence represented by SEQ ID NO: 7, encoding rat ZAQ2 containing the amino acid sequence represented by SEQ ID NO: 6 (Patent Document 2);
(2) A mouse mI5E (ZAQ2) gene (cDNA) encoding the mouse mI5E (ZAQ2) containing the amino acid sequence represented by SEQ ID NO: 8 and containing the nucleotide sequence represented by SEQ ID NO: 9 (see Patent Document 1) ) Is used.
[0011]
A mammalian ES cell in which the ZAQ gene has been inactivated refers to a gene that is artificially mutated in the ZAQ gene of the mammalian ES cell to suppress the gene expression ability or encode the gene. By substantially losing the activity of ZAQ, a mammalian ES cell in which the gene is substantially inactivated and has no ability to express ZAQ (hereinafter, may be referred to as a knockout gene of the present invention) can be obtained. Say.
In the present specification, examples of mammals used as ES cell materials include humans, cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and the like.
In the present specification, any non-human animal having a ZAQ gene may be used as the non-human animal, but a non-human mammal is preferable. As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among non-human mammals, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of diseased animal model systems, and are easy to breed, especially mice (for example, C57BL / 6 strain and DBA2 strain as pure strains) B6C3F ₁ System, BDF ₁ System, B6D2F ₁ Strains, BALB / c strains, ICR strains, etc. (preferably, pure strains such as C57BL / 6 strains, etc .; ₁ Strains or ICR strains)) or rats (eg, Wistar, SD, etc.) are particularly preferred.
[0012]
Examples of a method for artificially mutating the ZAQ gene include deletion of part or all of the gene sequence by genetic engineering techniques, or insertion or substitution of another gene. With these mutations, the knockout gene of the present invention can be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
Specific examples of mammalian (preferably non-human mammal) ES cells in which the ZAQ gene has been inactivated (hereinafter abbreviated as ZAQ gene inactivated ES cells or knockout ES cells) include, for example, drug resistance genes (Eg, a neomycin resistance gene, a hygromycin resistance gene or a zeocin resistance gene, preferably a neomycin resistance gene), or a reporter gene (eg, lacZ (Escherichia coli β-galactosidase gene), cat (chloramphenicol acetyltransferase gene) ), GUS (β-glucuronidase gene), luciferase gene, aequorin gene, taumarin gene, GFP (Green Fluorescent Protein) gene, etc., preferably lacZ etc.) are inserted. By destroying the exon function of the ZAQ gene, or by inserting a DNA sequence (for example, a polyA addition signal) that terminates the transcription of the gene into the introns between the exons, thereby making it impossible to synthesize complete mRNA. Then, a DNA vector (hereinafter, abbreviated as a targeting vector) having a DNA sequence constructed so as to disrupt the gene is produced. When a reporter gene is inserted to disrupt the function of an exon, the reporter gene is preferably inserted so as to be expressed under the control of the ZAQ promoter.
The “drug resistance gene” refers to a gene involved in drug resistance of antibiotics and the like, and is used as a marker for selecting whether or not the introduced gene has been expressed in cells.
The term "reporter gene" refers to a group of genes that serve as indicators of gene expression. Usually, structural genes of enzymes that catalyze a luminescence reaction or a color reaction are often used. (2) High sensitivity method capable of quantitatively expressing gene, (3) Low effect on transformed cells, (4) Localization of expression site And the like are preferably used (Plant Cell Engineering, Vol. 2, pp. 721, 1990). In addition, the above-mentioned “drug resistance gene” and the like are used for the same purpose, but the “reporter gene” is used to examine not only whether the gene to be introduced is expressed in cells but also in which tissue and when. And the expression level can be quantitatively and accurately examined.
Furthermore, a targeting vector is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are subjected to Southern hybridization analysis or DNA targeting on the targeting vector using a DNA sequence on or near the ZAQ gene as a probe. The knockout ES cells of the present invention can be selected by analyzing the sequence and the DNA sequence of the neighboring region other than the ZAQ gene used for the preparation of the targeting vector by PCR using primers.
Examples of the targeting vector include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTB5, pC194, etc.), and plasmids derived from yeast (eg, pSH19) PSH15), bacteriophages such as phage lambda, retroviruses such as Moloney leukemia virus, vaccinia virus or adenovirus vectors, baculovirus, bovine papillomavirus, viruses from the group of herpes viruses, or Epstein-Barr virus. Animal viruses and the like are used.
[0013]
As the original ES cells for inactivating the ZAQ gene by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known method of Evans and Kaufman. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2) ₁ ) Can also be used favorably. BDF ₁ In addition to the advantage that the number of eggs collected and the eggs are robust, the mouse has a C57BL / 6 mouse as a genetic background. , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced by C57BL / 6 mice.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, 8-cell stage embryos (8-cell stage embryos about 2.5 days after fertilization are preferable) ), A large number of early embryos can be obtained efficiently by culturing and using blastocysts.
[0014]
The secondary selection can be performed by confirming the number of chromosomes by the G-banding method or the like. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult to establish the cells due to physical operations or the like, the gene of the ES cells is knocked out, and then the normal cells (for example, the chromosome Is preferably 2n = 40).
The ES cell line obtained in this way usually has very good growth properties, but it must be carefully subcultured since it tends to lose its ontogenetic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably, 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas, 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on freshly prepared feeder cells. Such passage is usually performed every 1 to 3 days. At this time, cells are observed, and if morphologically abnormal cells are found, it is desired that the cultured cells be discarded.
ES cells can be differentiated into various cells such as parietal muscle, visceral muscle, myocardium, etc. by culturing ES cells in a monolayer up to high density under appropriate conditions or by culturing them in suspension until a cell clump is formed. Is possible [M. J. Evans and M.E. H. Kaufman, Nature, 292, 154, 1981; R. Martin Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], ZAQ gene expression-deficient cells obtained by differentiating the ES cells of the present invention can be obtained in vitro. Useful in cell biology studies of ZAQ.
When storing ES cells, use an appropriate freezing medium (eg, Dulbecco's modified Eagle's medium (DMEM) containing 10% DMSO and 10% fetal bovine serum) at about -80 ° C or lower. save.
[0015]
The non-human animal deficient in ZAQ gene expression of the present invention (hereinafter sometimes referred to as a non-human animal deficient in gene expression) may be, for example, a cell derived from a mammalian ES cell in which the ZAQ gene has been inactivated. It has been created by genetic engineering, and is, for example, a non-human animal in which germ cells and somatic cells have been introduced with an inactivated ZAQ gene sequence at an early stage of embryogenesis.
As the non-human animal, the same one as described above is used.
To knock out the ZAQ gene, the above-described targeting vector can be transferred to a non-human animal ES cell or a non-human animal egg cell by a known method (for example, electroporation, microinjection, calcium phosphate method, lipofection, aggregation, particle gun). , A DEAE-dextran method, etc. (preferred introduction methods include an electroporation method when introduced into ES cells and a microinjection method when introduced into egg cells). It can be carried out by replacing the inactivated ZAQ gene sequence with the ZAQ gene on the chromosome of a non-human animal ES cell or a non-human animal egg cell by homologous recombination.
Cells in which the ZAQ gene was knocked out were subjected to Southern hybridization analysis using a DNA sequence on or near the ZAQ gene as a probe, and a DNA sequence in a region near the ZAQ gene used in the targeting vector as a primer. It can be determined by analysis using the PCR method described above.
When non-human animal ES cells are used, a cell line in which the ZAQ gene has been inactivated is cloned by homologous recombination, and the cells are cloned at an appropriate time in the early stage of embryogenesis, for example, at the 8-cell stage. A chimeric embryo prepared by injecting into an animal embryo or blastocyst (injection method) or sandwiching the ES cell mass in which the ZAQ gene is inactivated with two 8-cell stage embryos (assembly chimera method) Implantation into the uterus of the pregnant non-human animal.
The created animal is a chimeric animal composed of both cells having a normal ZAQ locus and cells having an artificially mutated ZAQ locus.
When a part of the germ cells of the chimeric animal has a mutated ZAQ locus, all tissues were artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It is obtained by selecting individuals composed of cells having the ZAQ locus, for example, by judging coat color or the like. The individuals thus obtained are usually ZAQ heterozygous expression-deficient individuals, and cross-breeding of ZAQ heteroexpression-individuals can be performed to obtain ZAQ homoexpression-deficient individuals from their offspring.
When using an egg cell, for example, a transgenic non-human animal having a targeting vector introduced into a chromosome can be obtained by injecting a gene solution into a nucleus of an egg cell by a microinjection method, and these transgenic non-human animals can be obtained. Can be obtained by selecting those having a mutation in the ZAQ locus by homologous recombination.
A non-human animal deficient in ZAQ gene expression can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
[0016]
In the individual in which the ZAQ gene has been knocked out in this manner, animal individuals obtained by mating can confirm that the gene has been knocked out, and can be reared in a normal rearing environment.
Furthermore, the germline can be obtained and maintained according to a conventional method. That is, by mating male and female animals having the inactivated gene sequence, a homozygous animal having the inactivated gene sequence on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated gene sequence can be bred and subcultured. The progeny of the animal having the inactivated gene sequence thus obtained is also included in the non-human animal deficient in ZAQ gene expression of the present invention.
The non-human animal lacking ZAQ gene expression of the present invention exhibits the following phenotype.
(1) In a female animal, a fertilized egg cannot be stably obtained by superovulation.
(2) When fertilized eggs are cultured, the incidence of early embryos is low.
Such a mammalian ES cell in which the ZAQ gene has been inactivated is very useful for producing a non-human animal deficient in ZAQ gene expression. A non-human animal deficient in ZAQ gene expression; a disease model animal caused by drug induction or stress load on the animal; a better disease model animal produced by crossing a non-human animal deficient in ZAQ gene expression with another disease model animal; A disease model animal caused by drug induction or stress load on a disease model animal caused by crossing a non-human animal expression deficient animal with another disease model animal, and a bone marrow transplant animal using a non-human animal deficient in ZAQ gene expression and another disease model animal Or their tissues or cells derived therefrom, are diseases resulting from the deficiency of ZAQ, such as diseases resulting from inactivation of the biological activity of ZAQ based on the loss of various biological activities that can be induced by ZAQ. (Eg, digestive disorders (eg, enteritis, diarrhea, constipation, absorption Benign syndrome, etc.), endocrine disorders, adenosis, cancer, because it can be a better model of immunodeficiency or neurological disease), offers an effective study to investigate causes for and therapy for these diseases. Here, as another disease state model animal, for example, a model mouse using bone marrow transplantation limited to deficiency or elevation of gene expression only on the blood cell side is also mentioned [Linton, M. et al. F. , Et al. , Science 267: 1034-1037 (1995)]. Bone marrow transplanted mice have the potential to become more suitable disease models because of limited changes in gene function. For example, a mouse transplanted into another recipient animal whose bone marrow was collected and bone marrow was previously destroyed by irradiation, or a pathological model animal obtained from the above-described non-human animal lacking ZAQ gene expression as a donor animal, or Bone marrow transplanted mice transplanted as a recipient animal with a pathological model animal obtained from a non-human animal deficient in ZAQ gene expression in which bone marrow is collected and bone marrow is previously destroyed by irradiation with another pathological model animal as a donor animal are used. included.
Thus, the non-human animal deficient in ZAQ gene expression of the present invention, a pathological model animal caused by drug induction or stress load on the animal, a pathological model animal caused by crossing a non-human animal deficient in ZAQ gene expression with another pathological model animal A disease model animal caused by drug induction or stress load on a disease model animal caused by crossing a non-human animal lacking ZAQ gene expression with another disease model animal; a disease model animal obtained from a non-human animal lacking ZAQ gene expression; The pathological model animals obtained by bone marrow transplantation using the pathological model animals described above, or their tissues or cells derived therefrom, can be used for screening for preventive / therapeutic agents for the diseases. Here, examples of the above-mentioned tissue and cells derived therefrom include measuring specific activity using a homogenate such as liver or kidney, or measuring the activity or production amount of a specific product using peritoneal macrophages. It can be used for screening.
[0017]
[Screening method A of the present invention]
Non-human animal deficient in ZAQ gene expression used in the screening method of the present invention, a disease model animal caused by drug induction or stress load of the animal, a disease model produced by mating a non-human animal deficient in ZAQ gene expression with another disease model animal Animal, a disease model animal produced by crossing a non-human animal lacking ZAQ gene expression with another disease model animal, a disease model animal caused by drug induction or stress load on a disease model animal, and a disease model animal obtained from a non-human animal lacking ZAQ gene expression. The disease model animals obtained by bone marrow transplantation using other disease model animals, or their tissues or cells derived therefrom, include the same ones as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
Specifically, a non-human animal deficient in ZAQ gene expression of the present invention, a pathological model animal caused by drug induction or stress load of the animal, a pathological model generated by mating a non-human animal deficient in ZAQ gene expression with another pathological model animal Animal, a disease model animal caused by drug induction or stress loading on a disease model animal caused by crossing a non-human animal lacking ZAQ gene expression with another disease model animal, and a disease model animal obtained from a non-human animal lacking ZAQ gene expression. A pathological model animal obtained by bone marrow transplantation using another pathological model animal (hereinafter, sometimes referred to as a ZAQ gene expression-deficient non-human animal or the like), or a tissue or cells derived therefrom, is used as a test compound. , And compared with an untreated control animal, and each organ, tissue, cell, The change of such symptoms disease can be tested for prophylactic and therapeutic effect of the test compound as an indicator.
As a method for treating a test animal (a non-human animal deficient in ZAQ gene expression) with a test compound, for example, oral administration, intravenous injection and the like are used, and the method is appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like. be able to. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
For example, when screening for a prophylactic / therapeutic agent for enteritis, screening can be performed by administering a test compound to a non-human animal or the like deficient in expression of the ZAQ gene of the present invention and measuring the intestinal contraction of the animal over time. .
For example, in the screening method, when the test compound is administered to a test animal, the intestinal contraction of the test animal is reduced by about 10% or more, preferably about 30% or more, more preferably about 50% or more. Against diseases caused by inactivation of the biological activity of ZAQ (eg, gastrointestinal disorders (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.), gonadal disorders, cancer, immunodeficiency or neurological disorders) Can be selected as a substance having a therapeutic / preventive effect.
[0018]
The prophylactic / therapeutic agent obtained by using the screening method of the present invention contains a compound selected from the test compounds described above, and has a preventive / therapeutic effect for a disease caused by ZAQ deficiency. It is useful as a drug such as a safe and low toxic therapeutic / prophylactic drug for diseases caused by the disease. Further, a compound derived from the compound can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, and a salt with an organic acid. And pharmaceutically acceptable salts such as salts with basic or acidic amino acids.
Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt and ammonium salt.
Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine And salt.
Preferable examples of the salt with an inorganic acid include, for example, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like.
Preferred examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid And the like.
Preferable examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ortin and the like, and preferable examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like.
[0019]
When the compound or a salt thereof obtained by using the screening method of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means, for example, a sugar-coated tablet or capsule, if necessary, It can be used orally as elixirs, microcapsules and the like, or parenterally in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. For example, the compound or a salt thereof is admixed with pharmaceutically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in unit dosage form generally required for accepted pharmaceutical practice. Can be manufactured. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal 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, coconut oil and the like.
[0020]
Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents, For example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80) ^TM , HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol etc.), an antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered.
The dose of the compound or a salt thereof varies depending on symptoms and the like. However, in the case of oral administration, in general, for an adult (with a body weight of 60 kg) enteritis patient, about 0.1 to 100 mg per day, preferably It is about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. For example, in the case of an injectable form, usually in adult (as 60 kg) enteritis patients, It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0021]
[Screening method B of the present invention]
The present invention provides a method for screening a substance that promotes or inhibits the activity of a promoter for the gene of the present invention, which comprises administering a test compound to a non-human animal lacking ZAQ gene expression of the present invention and detecting the expression of a reporter gene. Provide a method.
In the above-mentioned screening method, the non-human animal deficient in expression of ZAQ gene of the present invention may be a non-human animal deficient in expression of ZAQ gene of the present invention in which the ZAQ gene is inactivated by introducing a reporter gene, A gene that can be expressed under the control of a promoter for the ZAQ gene is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a ZAQ gene expression-deficient non-human animal of the present invention in which the ZAQ gene is replaced with a reporter gene, since the reporter gene is under the control of a promoter for the ZAQ gene, by tracing the expression of a substance encoded by the reporter gene, The activity of the promoter can be detected.
For example, when a part of the ZAQ gene region is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, β-galactosidase is originally expressed instead of ZAQ in a tissue that expresses ZAQ. Therefore, for example, by staining with a reagent serving as a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), ZAQ can be easily prepared. The expression state in the animal body can be observed. Specifically, a ZAQ gene-deficient mouse or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or at about 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue sample with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
For example, in the screening method, when the test compound is administered to a test animal and the expression of the reporter protein is increased by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is expressed as ZAQ. When the test compound is administered to a test animal, the expression of the reporter protein is reduced by about 10% or more, preferably about 30% or more, more preferably about 50% or more. The test compound can be selected as a substance that inhibits the promoter activity for the ZAQ gene.
The substance obtained by using the above-mentioned screening method is a substance selected from the above-described test compounds, and is a substance that promotes or inhibits the promoter activity for the ZAQ gene.
The substance obtained by the screening method may form a salt. Examples of the salt of the substance include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
[0022]
Substances that promote or inhibit the promoter activity of the ZAQ gene can regulate the expression of ZAQ and regulate the function of ZAQ, for example, gastrointestinal diseases (enteritis, diarrhea, constipation, malabsorption syndrome, etc.) It can be used as a medicament such as an agent for preventing or treating diseases such as endocrine disease, gonadal disease, cancer, immunodeficiency or neurological disease.
Furthermore, substances derived from the substances obtained in the above screening can be used in the same manner.
[0023]
The drug containing the substance obtained by the screening method can be produced in the same manner as the drug containing the compound obtained by the above-mentioned screening method A.
The preparation obtained in this way is safe and low toxic, and thus can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the substance varies depending on the target disease, the administration subject, the administration route, and the like. For example, when a substance that inhibits the promoter activity of the ZAQ gene for the purpose of treating enteritis is orally administered, in general, an adult patient ( (With a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the substance are administered per day. When administered parenterally, the single dose of the substance varies depending on the administration subject, target disease, and the like. For example, a substance that promotes the promoter activity of the ZAQ gene for the purpose of treating enteritis may be in the form of an injection. Usually, when administered to an adult patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the substance per day is injected intravenously. It is convenient to administer. In the case of other animals, the amount can be administered in terms of 60 kg.
[0024]
As described above, the non-human animal deficient in ZAQ gene expression of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of a promoter for the ZAQ gene. Can greatly contribute to the investigation of the cause of the disease or the development of preventive and therapeutic drugs.
Also, using a DNA containing the promoter region of the ZAQ gene, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). For example, it is possible to specifically synthesize the peptide and examine its action in a living body. Furthermore, by linking an appropriate reporter gene to the above promoter portion and establishing a cell line in which this is expressed, a search system for a low molecular compound having an action of specifically promoting or suppressing the in vivo production ability of ZAQ itself. Can be used as
[0025]
In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or by abbreviations commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
RNA: ribonucleic acid
mRNA: messenger ribonucleic acid
Gly: glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: cysteine
Met: methionine
Glu: glutamic acid
Asp: Aspartic acid
Lys: lysine
Arg: Arginine
His: histidine
Phe: phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
[0026]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
2 shows the amino acid sequence of mouse-derived GPR73 (ZAQ1).
[SEQ ID NO: 2]
1 shows the nucleotide sequence of genomic DNA encoding mouse-derived GPR73 (ZAQ1).
[SEQ ID NO: 3]
2 shows the amino acid sequence of rat-derived ZAQ1.
[SEQ ID NO: 4]
1 shows the nucleotide sequence of cDNA encoding rat-derived ZAQ1.
[SEQ ID NO: 5]
1 shows the nucleotide sequence of cDNA encoding mouse-derived GPR73 (ZAQ1). [SEQ ID NO: 6]
2 shows the amino acid sequence of rat-derived ZAQ2.
[SEQ ID NO: 7]
1 shows the nucleotide sequence of cDNA encoding rat-derived ZAQ2.
[SEQ ID NO: 8]
2 shows the amino acid sequence of mouse-derived mI5E (ZAQ2).
[SEQ ID NO: 9]
1 shows the nucleotide sequence of cDNA encoding mouse-derived mI5E (ZAQ2).
[SEQ ID NO: 10]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 11]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 12]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 13]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 14]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 15]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 16]
3 shows the nucleotide sequence of a primer used in Example 3.
The transformant Escherichia coli JM109 / pKS-ZB obtained in Example 1 described below was obtained from October 1, 2002, at 1-1, Higashi 1-1, Tsukuba, Ibaraki, Japan, Central No. 6 (Zip code 305-8566). Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM BP-8215.
The transformant Escherichia coli JM109 / pZAQTgVP obtained in Example 2 to be described later has been administered by independent administration of 1-1, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan since October 17, 2002 (zip code 305-8566). Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-8216.
[0027]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.
[0028]
Example 1 Cloning of Mouse ZAQ1 Genomic DNA
Primer from exon 1 region of mouse GPR73
(32-54 ATACCACAGACACCTTTCACCGAC (SEQ ID NO: 10) and
458-436 TTAGTGGAGACGTAGAGGGAGAC (SEQ ID NO: 11)) was prepared, a 430 bp DNA fragment was obtained from the mouse genome by PCR, and the DIG probe was prepared after confirming the nucleotide sequence. Plaque hybridization was performed on a mouse 129SvEv genomic library (Stratagene), and three positive clones were obtained. A 11 kbp Bam HI fragment was subcloned from the results of Southern hybridization, and the restriction enzyme sites and the nucleotide sequence were determined. As a result, a genomic DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 consisting of exon 1 and exon 2 was obtained. (See FIG. 1). This genomic DNA encoded mouse ZAQ1 consisting of the amino acid sequence represented by SEQ ID NO: 1. Escherichia coli JM109 was transformed with plasmid pKS-ZB containing exon 1 and exon 2 to obtain JM109 / pKS-ZB.
[0029]
Example 2 Construction of targeting vector and production of ES homologous recombinant
A vector in which a 2 kbp HindIII fragment containing a promoter region and exon 1 was deleted from the restriction map of the genome was devised, and 1.1 kb upstream of exon 1 and 6.3 kb downstream of exon 1 (intron, exon 2 Neomycin resistance gene). The diphtheria toxin gene was used as a negative marker (see FIG. 1). Mouse 129SvEv-derived ES cells (AB2.2, 10) were charged with 25 μg of the ZAQ targeting vector under electroporation (Gene Pulser_Electroporation System, manufactured by Bio-Rad) at 230 V and 500 μF. ⁷ cells). Electroporation was performed twice, and neomycin selective culture was performed in a total of six 10 cm dishes. As a result, about 1200 neomycin-resistant colonies appeared after 6 days. About 400 neomycin-resistant colonies were picked up and cultured in a 96-well plate, a part of which was cultured for DNA extraction (24-well plate, without feeder cells), and the rest was frozen and stored. Southern analysis was performed on 340 neomycin-resistant strains. Southern analysis was performed using the BamHI-XhoI, 370 bp fragment (a part of exon 2; see FIG. 1) outside the 3 ′ used as the targeting vector as a probe, and hybridized to the BamHI-digested genome (for the wild type, In the homologous recombinant at 10.6 kb, bands are observed at 10.6 kb and 6.5 kb; see FIG. 1). As a result, a 6.5 kb band considered to be a homologous recombinant was found in 10 out of 340 strains. Ten types of ES homologous recombinants selected by primary screening (Southern analysis using a 3′-side probe) were raised from cryopreservation and cultured. Five of the ten strains showed good growth, and DNA was extracted from the five strains (No. 122, 163, 274, 287, 418) and confirmed by Southern analysis. As a result, as in the case of the primary screening, two bands of 10.6 and 6.5 kb were observed in all five strains (FIG. 2, left panel). Furthermore, Southern analysis (genomic Bam HI digestion) was performed using a 5 ′ probe (XhoI fragment 600 bp; see FIG. 1). As a result, two bands (10.6) showing homologous recombinants were observed in all five strains. , 4.1 kb) were observed (FIG. 2, right panel).
Karyotype analysis was performed on three types of ES homologous recombinants (Nos. 122, 287, and 418). When 50 karyotype analyzes were performed for each, No. The normal rates (2n = 40) for 122, 287, and 418 were 60%, 74%, and 66%, respectively, and were considered to have no effect on germline transfer.
ES homologous recombinant No. The plasmid pZAQTgVP contained in E. coli JM109 was transformed into E. coli JM109 to obtain E. coli JM109 / pZAQTgVP.
[0030]
Example 3 Preparation of knockout mouse
Homologous recombinant No. Microinjection into 122 C57BL / 6J strain mouse blastocysts was performed. The first injection was performed using 26 blastocysts, and the injected blastocysts were transplanted into pseudopregnant mouse oviducts obtained by crossing with vasectomized mice separately to make them pregnant. Six offspring were obtained, and five chimeric mice were obtained. Four of them were mice with a chimera rate of almost 100%. In the second injection experiment, 14 blastocysts were each injected. As a result, 6 offspring were obtained. Five chimeric mice were obtained as in the first experiment. Four were mice with almost 100% chimera rate.
No. For 122, a total of 10 chimeric mice were obtained. Male chimeric mice showing a chimera ratio of almost 100% were bred to C57BL / 6 strain female mice to confirm germline transfer in offspring and to obtain heterozygous mice.
By crossing the chimera with the C57BL / 6 strain mouse, 68 ES cell-derived mice were obtained. Genomic DNA was purified from the mouse tail, and the conditions for genotyping by PCR were examined first. As a result of the examination, the primers share the same MZ1 (GGAGCTCTGCTCCTCAGTGG (SEQ ID NO: 12)) on the 3 ′ side of the targeted region, and the 5 ′ side is used for wild type detection. 13)) The neomycin resistance gene sequence NE-1 (CCGCTCTCATTGCTCCAGCGG (SEQ ID NO: 14)) was used for mutant detection, and KOD-plus- (manufactured by Toyobo Co., Ltd.) was used as the polymerase. For the wild type, only a 400 bp fragment was detected, and for a hetero-deficient individual, a gene was determined by PCR designed to detect the 400 bp and 600 bp fragments. As a result, a hetero-deficient mouse could be obtained.
Next, gene deletion was confirmed by Southern hybridization. The genome was treated overnight at 37 ° C. using the restriction enzyme Bam HI. The probe can be amplified with a primer in the 5 'upstream region of ZAQ exon 1, Z22: 5'-CATTGTGTTGCTTCTGCCCTTTG-3' (SEQ ID NO: 15) and Z24: 5'-AGTGTCCAGGATGCCAATGTGTG-3 '(SEQ ID NO: 16). A 5 Kbp fragment was used and labeled using a PCR DIG probe synthesis kit (Roche). Hybridization was performed at 42 ° C. overnight. Washing was performed twice for 15 minutes in a solution in which 0.1% SDS was added to 0.1 × SSC at 65 ° C.
As a result, a 4.1 Kbp fragment derived from homologous recombination was confirmed to be as dense as the 10.6 Kbp fragment derived from the wild type. These individuals were found to be hetero-deficient mice. Since some of the individuals determined this time are used as mating parents for obtaining homo-deficient individuals, homo-deficient mice were obtained by mating male hetero-defective individuals and female hetero-deficient individuals that could be determined to be hetero-defective individuals.
[0031]
Example 4 Analysis of homo-deficient mice
Next, after weaning of the homo-deficient mice, changes in body weight at 6 to 18 weeks of age were examined. As a result, in the homo-deficient mice, both males and females tended to have a slightly lower body weight than the wild-type mice. The body weight of the female homo-deficient mouse became significantly lower than that of the wild-type mouse after 15 weeks of age. However, there was no significant difference in the weight of male homo-deficient mice. At the same time, the food intake at the age of 16 and 17 weeks in the homo-deficient mice was measured. As a result, there was no difference in the amount of food consumed at the age of 16 weeks between wild-type mice and homo-deficient mice. However, the intake of 17-week-old homozygous mice was significantly lower in both sexes than in wild-type mice.
[0032]
Example 5 Analysis of ovarian function in homo-deficient mice
PMSG (pregnant horse serum gonadotropin, manufactured by Teikoku Organ Co., Ltd.) was intraperitoneally administered to 11-week-old female wild-type mice and female homo-deficient mice at 13:00, respectively. At 11:00 on day 3, hCG (human chorionic gonadotropin) was intraperitoneally administered to the mouse at 5 IU. After the administration, the mice were co-resident with the male C57BL / 6J strain mouse in a one-to-one relationship. On the fourth day, at 9 o'clock, the vaginal glands of each female mouse were examined to confirm the mating. The female mice in which mating was confirmed were sacrificed by cervical dislocation, the oviducts were cut out, and fertilized eggs were collected according to a conventional method. After washing, the collected fertilized eggs were transferred to a fertilized egg culture medium (Brinster's BMOC-3 medium, manufactured by GIBCO), and the temperature was adjusted to 37 ° C. ₂ The cells were cultured in an incubator. The number of fertilized eggs obtained from each mouse was calculated. As a result, the number of ovulations per female wild-type mouse was 20 to 30 in most cases, and was relatively stable. On the other hand, the number of ovulations per female homo-deficient mouse ranged widely from 10 or less to 60 or more.
When the obtained fertilized eggs were cultured for 1 day by a conventional method, about 76% of the fertilized eggs obtained from the female wild-type mouse developed to a 2-cell stage embryo or a 4-cell stage embryo. On the other hand, fertilized eggs of female homozygous deficient mice that developed up to the 2-cell stage embryo or the 4-cell stage embryo were 53%. On day 4 of culture, about 36% of the female wild-type mouse-derived embryos developed up to the morula or blastocyst. It was about 15%.
[0033]
【The invention's effect】
The non-human animal ES cells in which the ZAQ gene has been inactivated according to the present invention are very useful for producing a non-human animal deficient in ZAQ gene expression.
Since the non-human animal lacking ZAQ expression of the present invention lacks various biological activities that can be induced by ZAQ, it can serve as a model for a disease caused by inactivation of the biological activity of ZAQ. It is useful for screening for prophylactic / therapeutic agents for various diseases, for investigating the causes of ZAQ-related diseases, and for examining treatment methods.
The ZAQ gene-introduced non-human animal of the present invention can elucidate the pathological mechanism of ZAQ-related diseases such as hyper-ZAQ disease and ZAQ dysfunction, and examine methods for treating these diseases. Further, it can be used for screening a therapeutic drug for the above ZAQ-related disease.
The screening method of the present invention can efficiently screen preventive / therapeutic agents for various diseases caused by ZAQ deficiency.
[0034]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the structures of a wild-type allele (upper panel), a ZAQ targeting vector (middle panel), and a homologous recombinant allele (lower panel).
Black boxes indicate ZAQ coding exons. The white box indicates the expression cassette of the foreign gene, and the arrow in the box indicates the direction of transcription. Neo indicates the neomycin resistance gene, and DTA indicates the diphtheria exotoxin gene.
FIG. 2 is a photograph showing the results of Southern analysis showing that the selected ES cell line is a homologous recombinant having a heterozygous deletion of ZAQ gene.
Genomic DNA was extracted from each of the five ES cell lines (No. 122, 163, 274, 287, 418) and the wild type (Wild), cut with Bam HI, and the 3′-side probe (see FIG. 1; left panel); Southern analysis was performed using the 5 'probe and the 5' probe (see Fig. 1; right panel). In the former, two bands of 10.6 and 6.5 kb were observed in all five strains, and in the latter, two bands of 10.6 and 4.1 kb were observed in all five strains. Five ES cell lines, 122, 163, 274, 287 and 418, were found to be homologous recombinants deficient in the ZAQ gene.

Claims (28)

A mammalian embryonic stem cell in which the ZAQ gene has been inactivated. The embryonic stem cell according to claim 1, which is drug resistant. The embryonic stem cell according to claim 2, wherein the drug is neomycin. The embryonic stem cell according to claim 1, wherein the ZAQ gene has been inactivated by inserting a reporter gene. The embryonic stem cell according to claim 4, wherein the reporter gene is a lacZ gene. The embryonic stem cell according to claim 1, wherein the mammal is a mouse. The embryonic stem cell according to claim 1, wherein the ZAQ gene is a gene containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. A non-human mammal deficient in ZAQ gene expression. The animal according to claim 8, wherein the ZAQ gene has been inactivated by the insertion of the reporter gene. The animal according to claim 9, wherein the reporter gene is under the control of a promoter for the ZAQ gene. The animal according to claim 8, wherein the non-human mammal is a mouse. The animal according to claim 8, wherein the ZAQ gene is a gene containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. As compared to the corresponding ZAQ gene-expressing animal,
(1) In a female animal, a fertilized egg cannot be stably obtained by superovulation treatment, and / or (2) a low incidence of early embryos when a fertilized egg is cultured.
The animal according to any one of claims 8 to 12, which has the phenotype: A method for screening a substance for preventing or treating a gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising using the animal according to claim 8 or a tissue thereof or a cell derived therefrom. 15. The screening method according to claim 14, wherein a test compound is administered to the animal according to claim 8, a tissue thereof, or cells derived therefrom. A substance for preventing or treating a digestive organ disease, endocrine disease, gonadal disease, cancer, immunodeficiency or neurological disease obtained by the screening method according to claim 14 or 15. A prophylactic / therapeutic agent for a digestive organ disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising the prophylactic / therapeutic substance according to claim 16. A disease model animal produced by crossing the animal according to claim 8 with another disease model animal. A disease model animal produced by drug induction or stress load on the animal according to claim 8. A disease model animal caused by drug induction or stress load on the animal according to claim 18. 21. Prevention and treatment of a digestive organ disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising using the animal according to any one of claims 18 to 20, or a tissue thereof, or a cell derived therefrom. A method for screening substances. A gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising administering a test compound to the animal according to any one of claims 18 to 20, or a tissue thereof, or a cell derived therefrom. Screening method for prophylactic and therapeutic substances. A prophylactic / therapeutic substance for digestive organ disease, endocrine disease, gonadal disease, cancer, immunodeficiency or neurological disease obtained by the screening method according to claim 21 or 22. A prophylactic / therapeutic agent for a digestive organ disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease, comprising the prophylactic / therapeutic substance according to claim 23. A method for screening a compound or a salt thereof that promotes or inhibits promoter activity on a ZAQ gene, comprising administering a test compound to the animal according to claim 10 and detecting expression of a reporter gene. A compound or a salt thereof that promotes or inhibits a promoter activity for a ZAQ gene obtainable by the screening method according to claim 25. A prophylactic / therapeutic treatment of a gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease comprising a compound or a salt thereof that promotes a promoter activity for a ZAQ gene obtainable by the screening method according to claim 25. Agent. A prophylactic / therapeutic treatment of a gastrointestinal disease, an endocrine disease, a gonadal disease, a cancer, an immunodeficiency or a neurological disease comprising a compound or a salt thereof that inhibits the promoter activity of the ZAQ gene obtainable by the screening method according to claim 25. Agent.

Images