WO2007080710A1 - Mouse deficient in vesicular gaba transporter gene - Google Patents

Mouse deficient in vesicular gaba transporter gene Download PDF

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WO2007080710A1
WO2007080710A1 PCT/JP2006/323297 JP2006323297W WO2007080710A1 WO 2007080710 A1 WO2007080710 A1 WO 2007080710A1 JP 2006323297 W JP2006323297 W JP 2006323297W WO 2007080710 A1 WO2007080710 A1 WO 2007080710A1
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mouse
gene
vgat
gaba transporter
deficient
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Yuchio Yanagawa
Kenji Nakamura
Minesuke Yokoyama
Shiro Konishi
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National University Corporation Gunma University
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy

Definitions

  • the present invention relates to a vesicular GABA transporter gene-deficient mouse useful as a model animal for cleft palate, umbilical hernia, central diseases and the like.
  • the brain is made up of a collection of neural networks composed of excitatory neurons and inhibitory neurons.
  • the major neurotransmitters (inhibitory neurotransmitters) of inhibitory neurons are ⁇ -aminobutyric acid (GABA) and glycine. Release GABA as a neurotransmitter Nerve cells are GABA neurons, which are widely distributed in the brain and spinal cord.
  • GABA neurons which are widely distributed in the brain and spinal cord.
  • neurons that release dalysin are glycine neurons, which are distributed mainly in the brain stem and spinal cord.
  • Inhibitory transmitters are known to play a central role in building brain functions such as arousal, sleep, circadian rhythm and learning, movement, and sensory information processing. .
  • GABA has been reported to be associated with neuropsychiatric disorders such as epilepsy and alcohol psychosis, and mental symptoms such as anxiety and depression.
  • GABA and GABA receptors has also been confirmed in non-neural tissues, suggesting that GABA neurotransmission functions outside the nervous system.
  • glycine is known to be associated with startle disease.
  • VGAT vesicle-type GABA transporter
  • Non-patent Document 1 a vesicle-type GABA transporter
  • VGAT is specifically expressed in inhibitory neurons (GABA neurons and glycine neurons), it is also used as a marker for inhibitory neurons.
  • no knockout mice of the VGAT gene have been reported, and the role of VGAT in vivo has not been fully elucidated.
  • Cleft palate is a state in which the palate (upper jaw) is born and tears.
  • the palate is formed by the fusion of the left and right palatal protrusions at the central part around the 7th to 12th weeks of pregnancy, but for some reason the palate part does not fuse well, and cleft palate may occur. is there. Many causes of cleft palate are unknown and related genes and model animals are also limited.
  • umbilical hernia refers to a state in which the intestinal tract or liver is wrapped in a hernia sac and escapes into the umbilical cord.
  • As a treatment method there is a method in which an organ is replaced by surgery and the abdominal wall is closed. Many causes of umbilical hernia are unknown, and related genes and model animals are limited.
  • Non-Patent Document 1 Nature. 1997 Oct 23; 389 (6653): 870_6.
  • An object of the present invention is to provide a model animal for central diseases caused by cleft palate, umbilical hernia, or nerve transmission through VGAT.
  • mice lacking the VGAT gene exhibit symptoms such as cleft palate and umbilical hernia.
  • the present invention has been completed.
  • the present invention is as follows.
  • a method for analyzing a central disease comprising using the mouse according to (7) or a nerve cell obtained from the mouse.
  • a screening method for a therapeutic drug for a central disease which comprises administering or supplementing a test compound to the mouse of (7) or a nerve cell obtained from the mouse.
  • FIG. 1 shows a procedure for inactivation of VGAT gene.
  • A shows the V GAT locus on the wild-type mouse chromosome
  • (b) shows the targeting vector
  • (c) shows the VGAT locus where homologous recombination with the targeting vector occurred
  • (d) Shows the VGAT locus after crossing with CAG-Cre mice.
  • FIG. 2 is a diagram (photograph) showing the results of Western method using anti-VGAT / 3 antibody. Wild type mice (1 ane 1), heterozygous VGAT gene-deficient mice (lanes 2 and 3), homozygous VGAT gene-completely deficient mice (lanes 4, 5) embryonic VGAT in the 18.5 day brain The figure which shows protein expression.
  • FIG. 3 A diagram (photograph) showing the palate of a wild-type mouse and a mouse completely deficient in the VGAT gene.
  • FIG. 4 A diagram (photograph) showing the front part of the abdominal wall of embryonic day 18.5 wild-type mice and VGAT gene-deficient mice.
  • the VGAT-deficient mouse of the present invention is a mouse in which the function of the VGAT protein is deficient by replacing the VGAT gene on the chromosome with an inactive VGAT gene.
  • “Inactive VGAT gene” refers to a gene that is unable to express normal VGAT protein due to partial deletion of the VGAT gene or insertion of another nucleotide sequence into the coding region of the VGAT gene. Examples of the defective VGAT gene include, but are not limited to, genes lacking exons 2 and 3.
  • VGAT protein ⁇ Deficient means that the function of VGAT protein such as GABA and glycine transport has been lost, and preferably means that the function of VGAT protein has been completely lost, as in hetero knockout mice. This includes cases in which only one of the alleles is replaced with an inactive type, and the function of the VGAT protein is partially lost.
  • the VGAT-deficient mouse of the present invention includes those in a fetal state.
  • Examples of the VGAT gene include a mouse VGAT gene having the base sequence represented by SEQ ID NO: 1. This base sequence is registered in GenBank with the accession number AB080232, and the information of the coding region can be obtained by referring to this number. Since the VGAT gene varies depending on the species, it may be the homologous gene of SEQ ID NO: 1. The homologous gene is homologous enough to cause homologous recombination with the VGAT gene on the mouse chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1. Those having homology are listed.
  • the homologous gene may be a gene that hybridizes with the gene having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.
  • stringent conditions for example, hybridization is performed, and washing is performed at 65 ° C, 1 X SSC, 0.1% SDS, preferably 65 ° C, 0.1 X SSC, 0.1% SDS. Conditions are mentioned.
  • the VGAT gene-deficient mouse of the present invention can be prepared by a known gene recombination method (gene targeting method). For example, it can be prepared as follows: First, a partial fragment of the VGAT gene is prepared, and a targeting vector for replacing the VGAT gene with a defective type is prepared.
  • a drug resistance gene for selection of recombinants, it is preferable to incorporate a drug resistance gene into the targeting vector.
  • a marker gene for drug selection neomycin resistance gene, noidalomycin B phosphotransferase gene, etc. can be used.
  • the Cre-loxP system (R. Kuhn et al. Science, 269, 1427-1429, 1995) and the FLP / FRT system (Rodriguez et al. Nat Genet 25: 139-40.) In that case, construct the targeting vector so that the partial sequence to be deleted is sandwiched between ⁇ sequences or FRT sequences.
  • a general method for obtaining a VGAT gene-deficient mouse using the above-described targeting vector is described below. However, the mouse of the present invention is not limited to that obtained by the following method.
  • Embryonic stem cells can be used for homologous recombination.
  • CCE cell line, TT2 cell line, 88_1 cell line, 1 cell line, R1 cell line, E14TG2a cell line, etc. can be used.
  • the targeting vector can be introduced into mouse ES cells according to a known method. For example, an electroporation method, a ribosome method, a calcium phosphate method, a DEAE-dextran method, and the like can be used.
  • a cell clone in which the wild-type VGAT gene on the chromosome is replaced with the mutant VGAT gene in the targeting vector is selected. Selection can be performed based on drug resistance, and it is preferable to confirm homologous recombination by Southern blotting, PCR, or the like.
  • ES cells having the mutated gene thus obtained are introduced into embryos of wild-type mice. Then, this ES cell-introduced embryo is transplanted into the uterus of a pseudo-parental foster parent mouse and allowed to give birth to produce a chimeric animal.
  • the force microinjection method in which the microinjection method and the aggregation method are known, is more preferable.
  • a pseudopregnant female mouse for use as a foster parent can be obtained by mating a normal-period female mouse with a male mouse castrated by vagina ligation or the like.
  • this chimeric mouse is mated with a pure-line mouse to produce a mouse derived from ES cells through the germ line.
  • a heterozygote deficient in the VGAT gene can be obtained by selecting an animal in which the recombinant ES cells transplanted into the embryo have entered the germline and breeding the animal.
  • VGAT gene-deficient homozygous mice By crossing the obtained VGAT gene-deficient heterozygote mice, VGAT gene-deficient homozygous mice can be obtained.
  • Cre / loxP system and FLP / FRT described above which are widely used in the production of a conditional knockout mouse, are used. It is also possible to use a system.
  • Cre recombination can be performed by mating with an animal expressing Cre recombinase, which is a recombination enzyme derived from P1 phage of E. coli, or by infecting a virus vector containing the Cre gene.
  • Cre / loxP system Cre recombination can be performed by mating with an animal expressing Cre recombinase, which is a recombination enzyme derived from P1 phage of E. coli, or by infecting a virus vector containing the Cre gene. Enzyme force
  • a VGAT gene-deficient mouse can be created to recognize and remove sequences sandwiched between xP sequences.
  • a knockout having the characteristic of tissue-specific gene deletion can be produced by mating with a mouse expressing tissue-specific Cre recombinase.
  • VGAT gene-deficient mice can be created by mating with animals that express FLP recombinase. It is also possible to obtain mice that are deficient in the VGAT gene in a time-specific manner by infecting a virus vector containing the Cre gene or the FLP gene with the power of mating with a mouse that expresses the recombinant enzyme or in a time-specific manner.
  • the VGAT gene-deficient mouse produced as described above exhibits cleft palate and umbilical hernia. Therefore, by using this mouse, it is possible to elucidate the onset mechanism of cleft palate and umbilical hernia and develop a treatment method.
  • VGAT gene-deficient mice can be used not only for these diseases but also for elucidating the onset mechanism of various diseases associated with VGAT.
  • diseases associated with VGAT include central diseases, particularly those involving inhibitory neurotransmission, and specifically include neuropsychiatric disorders such as epilepsy, alcoholism, anxiety, and depression. Disease.
  • neural tissue or nerve cells obtained from VGAT gene-deficient mice may be used.
  • VGAT gene-deficient mice can also be used for screening for therapeutic agents for the above diseases.
  • a therapeutic agent for the above-mentioned diseases can be screened by administering the compound to a VGAT gene-deficient mouse and evaluating the effect of improving the symptom of the compound.
  • the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel substances. It may be a known substance.
  • Examples of a method for treating the animal with a test substance include known methods commonly used such as oral administration and intravenous injection, and can be appropriately selected according to the symptom of the test animal, the nature of the test substance, and the like. Good.
  • the screening method of the present invention can also be performed using nerve cells or nerve tissue obtained from the genetically modified animal of the present invention.
  • VGAT knockout mice can examine the mechanism of epileptic seizures and anxiety in more detail by blocking inhibitory neurotransmission in a brain-specific and time-specific manner. In addition, many brain functions (emotion, breathing, circadian rhythm, etc.) are thought to play an important role in inhibitory neurotransmission, and VGAT knockout mice can also be used to elucidate brain function and its disorders. it can.
  • E. coli artificial chromosome (BAC) clones containing the VGAT gene were screened by PCR using a 129Sv mouse-derived BAC library (Genome system, St. Louis, MO, USA), and 3 clones were identified (Ebihara et al., Mol. Brain Res. 110, 126-139, 2003). These clones were purchased from the Genome system, purified BAC-derived DNA was digested with restriction enzymes, then incorporated into pBlueScript (+) (Stratagene), and the VGAT gene region was determined using the Southern method and sequence method. A genetic map was created ( Figure 1A). Prepared a cassette for PGK-neo gene disruption (Yanagawa et al., Transgenic Res.
  • the construct was inserted into the Kpnl site located 3 'of the VGAT gene so that the transcription direction was the same.
  • Another ⁇ site was inserted into the Xbal site in the first intron.
  • a targeting vector incorporating the diphtheria toxin gene for negative selection (Taniguchi et al., Neuron 19, 519-530, 1997) was obtained (Fig. Lb).
  • a targeting vector was introduced into ES cells using the electopore position method.
  • ES cells derived from 129Sv strain mice, A. Joyner et al. (Gene Targeting Second Edition: A Practical Approach. OXFORD University Press, New York, 200
  • cell culture and electoporation were performed.
  • G418-resistant transformants were cloned and cell lined.
  • FOG. Lc homologous recombinants
  • Homologous recombinant ES cells in which the targeting vector was integrated on the chromosome were injected into blastocyst stage embryos of C57BL / 6 mice.
  • a blastocyst stage embryo into which ES cells were injected was transplanted into a pseudo-parental foster mother mouse and allowed to give birth to a chimeric mouse.
  • This chimeric mouse was crossed with a wild type mouse to obtain a heterozygote. Next, it was crossed with a transgenic mouse that expresses Cre (CAG-Cre mouse: Sakai & Miyazaki, Biochem. Biophys. Res. Commun. 237, 318-24, 1997). Heterozygous mice (VGAT (+/-) mice) deficient in 3 exons were obtained (Fig. Id). Furthermore, this VGAT (+/-) mouse was crossed to obtain a homozygous VGAT gene-deficient mouse (VGAT (-/-) mouse).
  • VGAT protein expression wild-type mouse, VGAT (+/-) mouse, VGAT (-/-) mouse embryo 18.5 days old brain as a sample, anti-VGAT / 3 antibody (Takamori et al., J. Neurosci ⁇ , 20, 4904-4911, 2000).
  • VGAT (+/-) mice showed decreased expression of VGAT protein.
  • VGAT protein could not be detected (Fig. 2).
  • the VGAT gene-deficient mouse of the present invention can be used as a model animal for cleft palate and umbilical hernia to elucidate the onset mechanism of these diseases and develop therapeutic methods.

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Abstract

A mechanism for the development of cleft palate, omphalocele or a disease of the central nervous system can be elucidated and a therapeutic method and a therapeutic agent for the disease can be developed using a mouse deficient in a vesicular GABA transporter gene as a model animal.

Description

明 細 書  Specification
小胞性 GABAトランスポーター遺伝子欠損マウス  Vesicular GABA transporter gene-deficient mice
技術分野  Technical field
[0001] 本発明は、口蓋裂、臍帯ヘルニアや中枢性疾患などのモデル動物として有用な小 胞性 GABAトランスポーター遺伝子欠損マウスに関する。  [0001] The present invention relates to a vesicular GABA transporter gene-deficient mouse useful as a model animal for cleft palate, umbilical hernia, central diseases and the like.
背景技術  Background art
[0002] 脳は、興奮性ニューロンと抑制性ニューロンとで構成される神経ネットワークの集ま りからできてレ、る。抑制性ニューロンの主要な神経伝達物質 (抑制性神経伝達物質） は、 γ—ァミノ酪酸（GABA)とグリシンである。 GABAを神経伝達物質として放出する 神経細胞が GABAニューロンであり、脳および脊髄に広範囲に分布する。一方、ダリ シンを放出する神経細胞がグリシンニューロンであり、主に脳幹と脊髄に分布する。 抑制性伝達物質は神経の電位活動の制御を司り、覚醒、睡眠、概日リズムや学習、 運動、感覚情報処理など脳の機能を構築する上で中心的役割を果たしていることが 知られている。 GABAはてんかん病やアルコール精神病をはじめとする精神神経疾 患、不安や抑うつなどの精神症状との関連が報告されている。さらに、非神経組織で も GABAや GABA受容体の存在が確認されており、 GABA神経伝達は神経系以外で の機能も示唆されている。一方、グリシンはびつくり病（startle disease)との関連が知 られている。  The brain is made up of a collection of neural networks composed of excitatory neurons and inhibitory neurons. The major neurotransmitters (inhibitory neurotransmitters) of inhibitory neurons are γ-aminobutyric acid (GABA) and glycine. Release GABA as a neurotransmitter Nerve cells are GABA neurons, which are widely distributed in the brain and spinal cord. On the other hand, neurons that release dalysin are glycine neurons, which are distributed mainly in the brain stem and spinal cord. Inhibitory transmitters are known to play a central role in building brain functions such as arousal, sleep, circadian rhythm and learning, movement, and sensory information processing. . GABA has been reported to be associated with neuropsychiatric disorders such as epilepsy and alcohol psychosis, and mental symptoms such as anxiety and depression. In addition, the presence of GABA and GABA receptors has also been confirmed in non-neural tissues, suggesting that GABA neurotransmission functions outside the nervous system. On the other hand, glycine is known to be associated with startle disease.
従って、（l)GABAやグリシンの作用機序を明らかにすること、（2)GABAやグリシンを 介した神経伝達機能を明らかにすることは、脳や神経の機能を理解し、上記疾患の 発症機序の解明や治療法の確立に貢献できる。  Therefore, (l) elucidating the mechanism of action of GABA and glycine, (2) elucidating the neurotransmitter function via GABA and glycine, understanding the functions of the brain and nerves, and developing the above diseases Can contribute to elucidation of mechanisms and establishment of treatment methods.
GABAとグリシンは、小胞型 GABAトランスポーター (以下、 VGATと呼ぶ)によってシ ナプス小胞に蓄積された後に、シナプス間隙に放出される（非特許文献 1)。 VGAT は、抑制性ニューロン（GABAニューロンとグリシンニューロン）に特異的に発現するこ とから、抑制性ニューロンのマーカーとしても使用されている。しかしながら、 VGAT遺 伝子のノックアウトマウスは報告されておらず、生体内での VGATの役割はまだ十分 には解明されていない。 [0003] 口蓋裂は、口蓋（上あご）が生まれつき裂けてレ、る状態をレ、う。一般に、口蓋は、妊 娠 7週〜 12週目頃に左右の口蓋突起が中央部分で癒合することにより形成されるが 、何らかの理由により口蓋の部分がうまく癒合せず、 口蓋裂が生じることがある。 口蓋 裂の原因の多くは不明であり、関連遺伝子やモデル動物も限定されている。 GABA and glycine are accumulated in synaptic vesicles by a vesicle-type GABA transporter (hereinafter referred to as VGAT) and then released into the synaptic cleft (Non-patent Document 1). Since VGAT is specifically expressed in inhibitory neurons (GABA neurons and glycine neurons), it is also used as a marker for inhibitory neurons. However, no knockout mice of the VGAT gene have been reported, and the role of VGAT in vivo has not been fully elucidated. [0003] Cleft palate is a state in which the palate (upper jaw) is born and tears. In general, the palate is formed by the fusion of the left and right palatal protrusions at the central part around the 7th to 12th weeks of pregnancy, but for some reason the palate part does not fuse well, and cleft palate may occur. is there. Many causes of cleft palate are unknown and related genes and model animals are also limited.
一方、臍帯ヘルニアは、腸管や肝臓などがヘルニア嚢に包まれて臍帯中に脱出し ている状態をさす。治療法として手術により臓器を還納し、腹壁を閉鎖する方法があ る。臍帯ヘルニアの原因の多くは不明であり、関連遺伝子やモデル動物も限定され ている。  On the other hand, umbilical hernia refers to a state in which the intestinal tract or liver is wrapped in a hernia sac and escapes into the umbilical cord. As a treatment method, there is a method in which an organ is replaced by surgery and the abdominal wall is closed. Many causes of umbilical hernia are unknown, and related genes and model animals are limited.
非特許文献 1 : Nature. 1997 Oct 23;389(6653):870_6.  Non-Patent Document 1: Nature. 1997 Oct 23; 389 (6653): 870_6.
発明の開示  Disclosure of the invention
[0004] 本発明は口蓋裂、臍帯ヘルニアや VGATを介した神経伝達の障害による中枢性疾 患のモデル動物を提供することを課題とする。  [0004] An object of the present invention is to provide a model animal for central diseases caused by cleft palate, umbilical hernia, or nerve transmission through VGAT.
[0005] 本発明者らは上記課題を解決するために鋭意検討を行った。その結果、 VGAT遺 伝子を欠損させたマウスが、口蓋裂や臍帯ヘルニアなどの症状を呈することを見出し[0005] The present inventors diligently studied to solve the above problems. As a result, we found that mice lacking the VGAT gene exhibit symptoms such as cleft palate and umbilical hernia.
、本発明を完成するに至った。 The present invention has been completed.
[0006] すなわち、本発明は以下のとおりである。 That is, the present invention is as follows.
(1)染色体上の小胞性 GABAトランスポーター遺伝子が不活性型小胞性 GABAトラ ンスポーター遺伝子に置換されたことにより、小胞性 GABAトランスポータータンパク 質の機能が欠損した、小胞性 GABAトランスポーター遺伝子欠損マウス。  (1) Deletion of vesicular GABA transporter gene, which lacks the function of vesicular GABA transporter protein by replacing the vesicular GABA transporter gene on the chromosome with the inactive vesicular GABA transporter gene mouse.
(2)不活性型小胞性 GABAトランスポーター遺伝子力 ェクソン 2及びェクソン 3が欠 損した小胞性 GABAトランスポーター遺伝子である、 (1)の小胞性 GABAトランスポ 一ター遺伝子欠損マウス。  (2) Inactive vesicular GABA transporter gene strength The vesicular GABA transporter gene-deficient mouse according to (1), which is a vesicular GABA transporter gene lacking exon 2 and exon 3.
(3)口蓋裂のモデルマウスである、（1)又は（2)の小胞性 GABAトランスポーター遺 伝子欠損マウス。  (3) The vesicular GABA transporter gene-deficient mouse according to (1) or (2), which is a model mouse for cleft palate.
(4) (3)のマウスを用いることを特徴とする、 口蓋裂の解析方法。  (4) A method for analyzing cleft palate, characterized by using the mouse of (3).
(5)臍帯ヘルニアのモデルマウスである、 （1)又は（2)の小胞性 GABAトランスポー ター遺伝子欠損マウス。  (5) The vesicular GABA transporter gene-deficient mouse according to (1) or (2), which is a model mouse of umbilical hernia.
(6) (5)のマウスを用いることを特徴とする、臍帯ヘルニアの解析方法。 (7)中枢性疾患のモデルマウスである、（1)又は（2)の小胞性 GABAトランスポータ 一遺伝子欠損マウス。 (6) A method for analyzing umbilical hernia characterized by using the mouse of (5). (7) The vesicular GABA transporter monogene-deficient mouse according to (1) or (2), which is a model mouse for central diseases.
(8) (7)のマウス又は該マウスから得られる神経細胞を用いることを特徴とする、中枢 性疾患の解析方法。  (8) A method for analyzing a central disease, comprising using the mouse according to (7) or a nerve cell obtained from the mouse.
(9) (7)のマウス又は該マウスから得られる神経細胞に試験化合物を投与又は添カロ することを特徴とする、中枢性疾患の治療薬のスクリーニング方法。  (9) A screening method for a therapeutic drug for a central disease, which comprises administering or supplementing a test compound to the mouse of (7) or a nerve cell obtained from the mouse.
図面の簡単な説明  Brief Description of Drawings
[0007] [図 1]VGAT遺伝子の不活性化の手順を示す図。 （a)は野生型マウスの染色体上の V GAT遺伝子座を示し、 (b)はターゲッティングベクターを示し、 （c)はターゲッティング ベクターとの相同組換えが起こった VGAT遺伝子座を示し、 (d)は CAG-Creマウスと 掛け合わせた後の VGAT遺伝子座を示す。 B : BstEII, E : EcoRI、 H : HindIII、 K : KpnI、 X : XbaI0 [0007] FIG. 1 shows a procedure for inactivation of VGAT gene. (A) shows the V GAT locus on the wild-type mouse chromosome, (b) shows the targeting vector, (c) shows the VGAT locus where homologous recombination with the targeting vector occurred, (d) Shows the VGAT locus after crossing with CAG-Cre mice. B: BstEII, E: EcoRI, H: HindIII, K: KpnI, X: XbaI 0
[図 2]抗 VGAT/3抗体を用いたウェスタン法の結果を示す図（写真)。野生型マウス (1 ane 1)、ヘテロ接合体の VGAT遺伝子欠損マウス（lane 2， 3)、ホモ接合体の VGAT遺 伝子完全欠損マウス（lane 4, 5)の胎生 18.5日の脳での VGAT蛋白質の発現を示す 図。  FIG. 2 is a diagram (photograph) showing the results of Western method using anti-VGAT / 3 antibody. Wild type mice (1 ane 1), heterozygous VGAT gene-deficient mice (lanes 2 and 3), homozygous VGAT gene-completely deficient mice (lanes 4, 5) embryonic VGAT in the 18.5 day brain The figure which shows protein expression.
[図 3]出生日の野生型マウス及び VGAT遺伝子完全欠損マウスの口蓋を示す図（写 真)。  [Fig. 3] A diagram (photograph) showing the palate of a wild-type mouse and a mouse completely deficient in the VGAT gene.
[図 4]胎生 18.5日の野生型マウス及び VGAT遺伝子完全欠損マウスの腹壁前部を示 す図（写真)。  [Fig. 4] A diagram (photograph) showing the front part of the abdominal wall of embryonic day 18.5 wild-type mice and VGAT gene-deficient mice.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0008] 以下に本発明を詳しく説明する。 [0008] The present invention is described in detail below.
本発明の VGAT欠損マウス（VGATノックアウトマウス）は、染色体上の VGAT遺伝子 が不活性型 VGAT遺伝子に置換されたことにより、 VGATタンパク質の機能が欠損し たマウスである。 「不活性型 VGAT遺伝子」とは、 VGAT遺伝子の一部の欠損、 VGAT 遺伝子のコード領域への他の塩基配列の揷入などにより、正常な VGATタンパク質を 発現できない遺伝子をいう。欠損型 VGAT遺伝子としては、ェクソン 2及び 3が欠損し た遺伝子などが挙げられるが、これには限定されない。「VGATタンパク質の機能が 欠損した」とは、 GABAやグリシンの輸送などの VGATタンパク質の機能が失われたこ とをいい、好ましくは、 VGATタンパク質の機能が完全に失われたことを意味するが、 ヘテロノックアウトマウスのように片方のアレルのみ不活性型に置換されて、 VGATタ ンパク質の機能が一部失われたような場合も含む。 The VGAT-deficient mouse of the present invention (VGAT knockout mouse) is a mouse in which the function of the VGAT protein is deficient by replacing the VGAT gene on the chromosome with an inactive VGAT gene. “Inactive VGAT gene” refers to a gene that is unable to express normal VGAT protein due to partial deletion of the VGAT gene or insertion of another nucleotide sequence into the coding region of the VGAT gene. Examples of the defective VGAT gene include, but are not limited to, genes lacking exons 2 and 3. “The function of VGAT protein `` Deficient '' means that the function of VGAT protein such as GABA and glycine transport has been lost, and preferably means that the function of VGAT protein has been completely lost, as in hetero knockout mice. This includes cases in which only one of the alleles is replaced with an inactive type, and the function of the VGAT protein is partially lost.
なお、本発明の VGAT欠損マウスは胎児の状態にあるものも含む。  The VGAT-deficient mouse of the present invention includes those in a fetal state.
[0009] VGAT遺伝子としては、例えば、配列番号 1で示される塩基配列を有するマウス VG AT遺伝子を挙げることができる。この塩基配列は、 GenBankにァクセッションナンパ 一 AB080232で登録されており、コード領域ゃェクソンの情報はこの番号を参照する ことによって得ることができる。なお、 VGAT遺伝子は、種によっても異なるため、配列 番号 1のホモログ遺伝子であってもよい。ホモログ遺伝子は、マウスの染色体上の VG AT遺伝子と相同組換えを起こしうる程度の相同性、例えば、配列番号 1の塩基配列 と 80%以上、好ましくは 90%以上、より好ましくは 95%以上の相同性を有するものが 挙げられる。また、ホモログ遺伝子は配列番号 1の塩基配列を有する遺伝子とストリン ジェントな条件下でハイブリダィズする遺伝子であってもよい。ここで、ストリンジェント な条件としては、例えば、ハイブリダィズを行レ、、 65°C、 1 X SSC, 0.1% SDS、好ま しくは、 65°C、 0.1 X SSC, 0.1% SDSの条件で洗浄する条件が挙げられる。  [0009] Examples of the VGAT gene include a mouse VGAT gene having the base sequence represented by SEQ ID NO: 1. This base sequence is registered in GenBank with the accession number AB080232, and the information of the coding region can be obtained by referring to this number. Since the VGAT gene varies depending on the species, it may be the homologous gene of SEQ ID NO: 1. The homologous gene is homologous enough to cause homologous recombination with the VGAT gene on the mouse chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1. Those having homology are listed. The homologous gene may be a gene that hybridizes with the gene having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions. Here, as stringent conditions, for example, hybridization is performed, and washing is performed at 65 ° C, 1 X SSC, 0.1% SDS, preferably 65 ° C, 0.1 X SSC, 0.1% SDS. Conditions are mentioned.
[0010] 本発明の VGAT遺伝子欠損マウスは、公知の遺伝子組み換え法（ジーンターゲッテ イング法）により作製することができる。例えば、以下のようにして作製することができる 先ず、 VGAT遺伝子の部分断片を用意し、 VGAT遺伝子を欠損型に置換するため のターゲテイングベクターを作製する。  [0010] The VGAT gene-deficient mouse of the present invention can be prepared by a known gene recombination method (gene targeting method). For example, it can be prepared as follows: First, a partial fragment of the VGAT gene is prepared, and a targeting vector for replacing the VGAT gene with a defective type is prepared.
組み換え体の選別のため、ターグティングベクターには薬剤耐性遺伝子を組み込 むことが好ましい。薬剤選択のマーカー遺伝子として、ネオマイシン耐性遺伝子、ノヽ イダロマイシン Bホスホトランスフェラーゼ遺伝子等を使用することができる。  For selection of recombinants, it is preferable to incorporate a drug resistance gene into the targeting vector. As a marker gene for drug selection, neomycin resistance gene, noidalomycin B phosphotransferase gene, etc. can be used.
また、 VGAT遺伝子の部分配列を欠損させるために Cre-loxPのシステム（R. Kuhn e t al. Science, 269,1427-1429, 1995)や FLP/FRTのシステム（Rodriguez et al. Nat G enet 25:139-40.)を用いてもよレ、。その場合、欠損させる部分配列が ΙοχΡ配列あるい は FRT配列の間に挟まれるようにターゲティングベクターを構築する。 [0011] 以下に、上記のようなターゲテイングベクターを用いて VGAT遺伝子欠損マウスを得 るための一般的な方法について述べる。ただし、本発明のマウスは以下の方法により 得られるものには限定されない。 In addition, the Cre-loxP system (R. Kuhn et al. Science, 269, 1427-1429, 1995) and the FLP / FRT system (Rodriguez et al. Nat Genet 25: 139-40.) In that case, construct the targeting vector so that the partial sequence to be deleted is sandwiched between ΙοχΡ sequences or FRT sequences. [0011] A general method for obtaining a VGAT gene-deficient mouse using the above-described targeting vector is described below. However, the mouse of the present invention is not limited to that obtained by the following method.
上記の方法により作製したターゲッティングベクターを使用して、相同組み換えを行 う。相同組換えには胚性幹細胞（ES細胞）を用いることができる。現在マウス由来の E S細胞株がいくつか確立されており、 CCE細胞株、 TT2細胞株、八8_ 1細胞株、】1 細胞株、 R1細胞株、 E14TG2a細胞株等を使用することができる。ターグティングべ クタ一は公知の方法に準じてマウス ES細胞に導入することができる。例えば、エレク トロポレーシヨン法、リボソーム法、リン酸カルシウム法、 DEAE—デキストラン法等が 利用できる。  Homologous recombination is performed using the targeting vector prepared by the above method. Embryonic stem cells (ES cells) can be used for homologous recombination. Several mouse ES cell lines have been established, and CCE cell line, TT2 cell line, 88_1 cell line, 1 cell line, R1 cell line, E14TG2a cell line, etc. can be used. The targeting vector can be introduced into mouse ES cells according to a known method. For example, an electroporation method, a ribosome method, a calcium phosphate method, a DEAE-dextran method, and the like can be used.
次いで、染色体上の野生型 VGAT遺伝子がターゲティングベクター中の変異 VGAT 遺伝子に置換された細胞クローンを選択する。選択は薬剤耐性などに基づレ、て行う ことができ、さらに、サザンブロッテイングや PCRなどにより相同組み換えを確認するこ とが好ましい。  Next, a cell clone in which the wild-type VGAT gene on the chromosome is replaced with the mutant VGAT gene in the targeting vector is selected. Selection can be performed based on drug resistance, and it is preferable to confirm homologous recombination by Southern blotting, PCR, or the like.
[0012] こうして得た変異遺伝子を持つ ES細胞を、野生型マウスの胚に導入する。そして、 この ES細胞導入胚を偽妊娠状態の仮親マウスの子宮に移植し、出産させることによ りキメラ動物を作製することができる。 ES細胞を胚盤胞期胚等の胚に導入する方法と しては、マイクロインジュクシヨン法や凝集法が知られている力 マイクロインジュクショ ン法がより好ましい。仮親とするための偽妊娠雌マウスは、正常性周期の雌マウスを、 精管結紮などにより去勢した雄マウスと交配することにより得ることができる。  [0012] ES cells having the mutated gene thus obtained are introduced into embryos of wild-type mice. Then, this ES cell-introduced embryo is transplanted into the uterus of a pseudo-parental foster parent mouse and allowed to give birth to produce a chimeric animal. As a method for introducing ES cells into embryos such as blastocyst stage embryos, the force microinjection method, in which the microinjection method and the aggregation method are known, is more preferable. A pseudopregnant female mouse for use as a foster parent can be obtained by mating a normal-period female mouse with a male mouse castrated by vagina ligation or the like.
[0013] 次いで、このキメラマウスを純系のマウスと交配し、生殖系列を通して ES細胞に由 来するマウスを作製する。胚内に移植された組み換え ES細胞が生殖系列に移行し た動物を選択し、その動物を繁殖させることにより、 VGAT遺伝子を欠損したヘテロ接 合体を得ることができる。  [0013] Next, this chimeric mouse is mated with a pure-line mouse to produce a mouse derived from ES cells through the germ line. A heterozygote deficient in the VGAT gene can be obtained by selecting an animal in which the recombinant ES cells transplanted into the embryo have entered the germline and breeding the animal.
得られた VGAT遺伝子欠損へテロ接合体マウス同士を交配させることにより、 VGAT 遺伝子欠損ホモ接合マウスを得ることができる。  By crossing the obtained VGAT gene-deficient heterozygote mice, VGAT gene-deficient homozygous mice can be obtained.
[0014] なお、本発明のノックアウトマウスを作製するにあたり、コンデイショナルなノックァゥ トマウスの作製において汎用されている、上述の Cre/loxPのシステムや FLP/FRTの システムを用いることも可能である。 Cre/loxPのシステムを用いる場合、大腸菌の P1 ファージ由来の組み換え酵素である Cre組換え酵素を発現している動物と交配させる か、または Cre遺伝子を含むウィルスベクターを感染させることにより、 Cre組換え酵素 力 xP配列で挟まれた配列を認識して除去するため、 VGAT遺伝子欠損マウスを作 出することができる。また、組織特異的に Cre組換え酵素を発現しているマウスと交配 させることにより、組織特異的に遺伝子が欠損した特性を有するノックアウトを作製す ることもできる。同様に、 FLP/FRTのシステムを用いる場合は、 FLP組換え酵素を発 現する動物と交配させることにより、 VGAT遺伝子欠損マウスを作出することができる また、時期特異的に Cre組換え酵素や FLP組換え酵素を発現するマウスと交配させ る力、あるいは時期特異的に Cre遺伝子または FLP遺伝子を含むウィルスベクターを 感染させることにより、時期特異的に VGAT遺伝子が欠損するマウスを得ることもでき る。 [0014] It should be noted that, in producing the knockout mouse of the present invention, the Cre / loxP system and FLP / FRT described above, which are widely used in the production of a conditional knockout mouse, are used. It is also possible to use a system. When using the Cre / loxP system, Cre recombination can be performed by mating with an animal expressing Cre recombinase, which is a recombination enzyme derived from P1 phage of E. coli, or by infecting a virus vector containing the Cre gene. Enzyme force A VGAT gene-deficient mouse can be created to recognize and remove sequences sandwiched between xP sequences. In addition, a knockout having the characteristic of tissue-specific gene deletion can be produced by mating with a mouse expressing tissue-specific Cre recombinase. Similarly, when the FLP / FRT system is used, VGAT gene-deficient mice can be created by mating with animals that express FLP recombinase. It is also possible to obtain mice that are deficient in the VGAT gene in a time-specific manner by infecting a virus vector containing the Cre gene or the FLP gene with the power of mating with a mouse that expresses the recombinant enzyme or in a time-specific manner.
[0015] 上記のようにして作製された VGAT遺伝子欠損マウスは、 口蓋裂や臍帯ヘルニアを 呈する。したがって、このマウスを用いることにより、口蓋裂や臍帯ヘルニアの発症メ 力二ズムの解明や治療法の開発を行うことができる。  [0015] The VGAT gene-deficient mouse produced as described above exhibits cleft palate and umbilical hernia. Therefore, by using this mouse, it is possible to elucidate the onset mechanism of cleft palate and umbilical hernia and develop a treatment method.
また、 口蓋裂や臍帯ヘルニアをもつヒトの遺伝子解析をする際に、 VGAT遺伝子を 原因の候補遺伝子として、その遺伝子変異などを解析することも可能である。特に、 家族性口蓋裂や臍帯ヘルニアでは重要な情報になる。  It is also possible to analyze the genetic variation of humans with cleft palate and umbilical hernia using VGAT gene as a candidate gene. This is especially important for familial cleft palate and umbilical hernia.
[0016] また、 VGAT遺伝子欠損マウスはこれらの疾患に限らず、 VGATが関連する各種疾 患の発症メカニズムの解明にも利用することができる。 VGATが関連する疾患として は、例えば、中枢性疾患、特に抑制性神経伝達が関与する中枢性疾患が挙げられ、 具体的には、てんかん病、アルコール依存症、不安症、抑鬱症などの精神神経疾患 が挙げられる。解析には、 VGAT遺伝子欠損マウスから得られる神経組織や神経細 胞を用いてもよい。  [0016] In addition, VGAT gene-deficient mice can be used not only for these diseases but also for elucidating the onset mechanism of various diseases associated with VGAT. Examples of diseases associated with VGAT include central diseases, particularly those involving inhibitory neurotransmission, and specifically include neuropsychiatric disorders such as epilepsy, alcoholism, anxiety, and depression. Disease. For the analysis, neural tissue or nerve cells obtained from VGAT gene-deficient mice may be used.
また、 VGAT遺伝子欠損マウスは上記疾患の治療薬のスクリーニングにも用いること ができる。例えば、 VGAT遺伝子欠損マウスに化合物を投与し、化合物の症状改善 効果を評価することにより、上記疾患の治療薬をスクリーニングすることができる。 被検物質としては、例えば、ペプチド、タンパク、非ペプチド性化合物、合成化合物 、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液等が挙げられ、これらの 物質は新規な物質であってもよいし、公知の物質であってもよレ、。該動物を被検物質 で処理する方法としては、例えば、経口投与、静脈注射等の通常用いられる公知の 方法が挙げられ、試験動物の症状、被検物質の性質等に合わせて適宜選択すれば よい。また、本発明のスクリーニング方法は、本発明の遺伝子改変動物から得られる 神経細胞や神経組織を用いて行うこともできる。 In addition, VGAT gene-deficient mice can also be used for screening for therapeutic agents for the above diseases. For example, a therapeutic agent for the above-mentioned diseases can be screened by administering the compound to a VGAT gene-deficient mouse and evaluating the effect of improving the symptom of the compound. Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel substances. It may be a known substance. Examples of a method for treating the animal with a test substance include known methods commonly used such as oral administration and intravenous injection, and can be appropriately selected according to the symptom of the test animal, the nature of the test substance, and the like. Good. The screening method of the present invention can also be performed using nerve cells or nerve tissue obtained from the genetically modified animal of the present invention.
条件付き VGATノックアウトマウスは、脳の部位特異的および時間特異的に抑制性 神経伝達を遮断することで、てんかん発作や不安症の発症機序をより詳細に検討す ることができる。さらに、多くの脳の機能（情動、呼吸、 日内リズム等）は抑制性神経伝 達が重要な役割を果たすと考えられており、 VGATノックアウトマウスは脳機能および その障害を解明することにも利用できる。  Conditional VGAT knockout mice can examine the mechanism of epileptic seizures and anxiety in more detail by blocking inhibitory neurotransmission in a brain-specific and time-specific manner. In addition, many brain functions (emotion, breathing, circadian rhythm, etc.) are thought to play an important role in inhibitory neurotransmission, and VGAT knockout mice can also be used to elucidate brain function and its disorders. it can.
実施例 Example
VGAT遺伝子を含む大腸菌人工染色体（BAC)クローンを 129Sv系統マウス由来 BA Cライブラリー（Genome system社、 St. Louis, MO, U.S.A.)を PCR法でスクリーニング し、 3クローン同定した (Ebihara et al.， Mol. Brain Res. 110, 126-139, 2003)。これらの クローンを Genome system社から購入し、精製した BAC由来の DNAを制限酵素で消 ィ匕した後に pBlueScript(+) (Stratagene)に組み込み、サザン法とシークェンス法を用 レ、て VGAT遺伝子領域を含む遺伝子地図を作成した（図 1A)。 PGK-neo遺伝子破壊 用カセット（Yanagawa et al.， Transgenic Res. 8, 215-221, 1999)を用意し、その前後 に FRTサイトを、 3'側に ΙοχΡサイトを連結することにより得られた DNA構築物を VGAT 遺伝子の 3'側に存在する Kpnlサイトに転写の向きが同じになるように挿入した。もう 一つの ΙοχΡサイトを第 1イントロンに存在する Xbalサイトに挿入した。さらに、ネガティ ブ選択用のジフテリア毒素遺伝子（Taniguchi et al.， Neuron 19， 519-530, 1997)を 組み込んだターゲッティングベクターを得た（図 lb)。  E. coli artificial chromosome (BAC) clones containing the VGAT gene were screened by PCR using a 129Sv mouse-derived BAC library (Genome system, St. Louis, MO, USA), and 3 clones were identified (Ebihara et al., Mol. Brain Res. 110, 126-139, 2003). These clones were purchased from the Genome system, purified BAC-derived DNA was digested with restriction enzymes, then incorporated into pBlueScript (+) (Stratagene), and the VGAT gene region was determined using the Southern method and sequence method. A genetic map was created (Figure 1A). Prepared a cassette for PGK-neo gene disruption (Yanagawa et al., Transgenic Res. 8, 215-221, 1999). DNA obtained by linking FRT sites before and after it and ΙοχΡ sites on the 3 'side. The construct was inserted into the Kpnl site located 3 'of the VGAT gene so that the transcription direction was the same. Another ΙοχΡ site was inserted into the Xbal site in the first intron. Furthermore, a targeting vector incorporating the diphtheria toxin gene for negative selection (Taniguchi et al., Neuron 19, 519-530, 1997) was obtained (Fig. Lb).
次にエレクト口ポレーシヨン法を用いて ES細胞にターゲッティングベクターを導入し た。 129Sv系統マウスに由来する ES細胞を使用し、 A.し Joyner et al. (Gene Targetin g Second Edition: A Practical Approach. OXFORD University Press, New York, 200 0)に記載された方法にしたがって、細胞培養及びエレクト口ポレーシヨンを行った。タ ーゲティングベクターを導入した ES細胞をジヱネティシン（G418)含有培地で培養す ることにより、 G418耐性形質転換体をクローユングしてセルライン化した。つづいて、 ターグティングベクターによる相同組換え体（図 lc)をサザン法によって確認した。 ターゲテイングベクターが染色体上に組み込まれた相同組換え体 ES細胞を C57BL /6マウスの胚盤胞期胚に注入した。偽妊娠させた仮親マウスに、 ES細胞を注入した 胚盤胞期胚を移植し、出産させてキメラマウスを得た。 Next, a targeting vector was introduced into ES cells using the electopore position method. Using ES cells derived from 129Sv strain mice, A. Joyner et al. (Gene Targeting Second Edition: A Practical Approach. OXFORD University Press, New York, 200 In accordance with the method described in 0), cell culture and electoporation were performed. By culturing ES cells introduced with the targeting vector in a medium containing geneticin (G418), G418-resistant transformants were cloned and cell lined. Subsequently, homologous recombinants (FIG. Lc) using the targeting vector were confirmed by the Southern method. Homologous recombinant ES cells in which the targeting vector was integrated on the chromosome were injected into blastocyst stage embryos of C57BL / 6 mice. A blastocyst stage embryo into which ES cells were injected was transplanted into a pseudo-parental foster mother mouse and allowed to give birth to a chimeric mouse.
このキメラマウスを野生型マウスと交配してヘテロ接合体を得た。次に Creを発現す るトランスジエニックマウス（CAG- Creマウス： Sakai & Miyazaki, Biochem. Biophys. Re s. Commun. 237, 318-24, 1997)と掛け合わせて、 VGAT遺伝子の第 2、第 3ェクソン が欠損したヘテロ接合体マウス（VGAT (+/-)マウス）を得た（図 Id)。さらにこの VGAT (+/-)マウスを掛け合わせて、ホモ接合体の VGAT遺伝子完全欠損マウス (VGAT (- /-)マウス)を得た。  This chimeric mouse was crossed with a wild type mouse to obtain a heterozygote. Next, it was crossed with a transgenic mouse that expresses Cre (CAG-Cre mouse: Sakai & Miyazaki, Biochem. Biophys. Res. Commun. 237, 318-24, 1997). Heterozygous mice (VGAT (+/-) mice) deficient in 3 exons were obtained (Fig. Id). Furthermore, this VGAT (+/-) mouse was crossed to obtain a homozygous VGAT gene-deficient mouse (VGAT (-/-) mouse).
VGAT蛋白質の発現にっレ、て野生型マウス、 VGAT (+/-)マウス、 VGAT (-/-)マウ スの胎生 18.5日の脳をサンプルとして、抗 VGAT/3抗体（Takamori et al., J. Neurosci ·, 20， 4904-4911, 2000)を用いたウェスタン法で検討した。野生型マウスに比較して VGAT (+/-)マウスでは VGAT蛋白質の発現量が減少してレ、た。 VGAT (-/-)マウス では、 VGAT蛋白質が検出できな力 た（図 2)。  VGAT protein expression, wild-type mouse, VGAT (+/-) mouse, VGAT (-/-) mouse embryo 18.5 days old brain as a sample, anti-VGAT / 3 antibody (Takamori et al., J. Neurosci ·, 20, 4904-4911, 2000). Compared to wild-type mice, VGAT (+/-) mice showed decreased expression of VGAT protein. In VGAT (-/-) mice, VGAT protein could not be detected (Fig. 2).
この VGAT遺伝子完全欠損マウス（ホモ接合体）について調べた結果、口蓋裂（図 3 )、臍帯ヘルニア（図 4)を呈しており、出生日に死亡を観察した。  As a result of investigating this VGAT gene complete deficiency mouse (homozygote), cleft palate (Fig. 3) and umbilical hernia (Fig. 4) were observed, and death was observed on the day of birth.
産業上の利用可能性 Industrial applicability
本発明の VGAT遺伝子欠損マウスは口蓋裂や臍帯ヘルニアのモデル動物としてこ れらの疾患の発症機序の解明や治療法の開発に使用することができる。また、脳機 能における抑制性神経伝達の役割の解明とそれに基づく薬剤の開発、不安、抑鬱、 てんかん、神経細胞死などの抑制性神経伝達が関与する中枢性疾患の検索とそれ に基づく薬剤の開発にも使用することができる。  The VGAT gene-deficient mouse of the present invention can be used as a model animal for cleft palate and umbilical hernia to elucidate the onset mechanism of these diseases and develop therapeutic methods. In addition, elucidation of the role of inhibitory neurotransmission in brain function and development of drugs based on it, search for central diseases involving inhibitory neurotransmission such as anxiety, depression, epilepsy, and neuronal cell death, It can also be used for development.

Claims

請求の範囲 The scope of the claims
[1] 染色体上の小胞性 GABAトランスポーター遺伝子が不活性型小胞性 GABAトランス ポーター遺伝子に置換されたことにより、小胞性 GABAトランスポータータンパク質 の機能が欠損した、小胞性 GABAトランスポーター遺伝子欠損マウス。  [1] A vesicular GABA transporter gene-deficient mouse in which the function of the vesicular GABA transporter protein is deleted by replacing the vesicular GABA transporter gene on the chromosome with an inactive vesicular GABA transporter gene.
[2] 不活性型小胞性 GABAトランスポーター遺伝子力 ェクソン 2及びェクソン 3が欠損 した小胞性 GABAトランスポーター遺伝子である、請求項 1に記載の小胞性 GABA トランスポーター遺伝子欠損マウス。 [2] The vesicular GABA transporter gene-deficient mouse according to claim 1, which is a vesicular GABA transporter gene deficient in inactive vesicular GABA transporter gene exon 2 and exon 3.
[3] 口蓋裂のモデルマウスである、請求項 1又は 2に記載の小胞性 GABAトランスポータ 一遺伝子欠損マウス。 [3] The vesicular GABA transporter single gene-deficient mouse according to claim 1 or 2, which is a model mouse for cleft palate.
[4] 請求項 3に記載のマウスを用いることを特徴とする、 口蓋裂の解析方法。 [4] A method for analyzing cleft palate, wherein the mouse according to claim 3 is used.
[5] 臍帯ヘルニアのモデルマウスである、請求項 1又は 2に記載の小胞性 GABAトランス ポーター遺伝子欠損マウス。 [5] The vesicular GABA transporter gene-deficient mouse according to claim 1 or 2, which is a umbilical hernia model mouse.
[6] 請求項 5に記載のマウスを用レ、ることを特徴とする、臍帯ヘルニアの解析方法。 [6] A method for analyzing umbilical hernia, wherein the mouse according to claim 5 is used.
[7] 中枢性疾患のモデルマウスである、請求項 1又は 2に記載の小胞性 GABAトランスポ 一ター遺伝子欠損マウス。 [7] The vesicular GABA transporter gene-deficient mouse according to claim 1 or 2, which is a model mouse for central diseases.
[8] 請求項 7に記載のマウス又は該マウスから得られる神経細胞を用いることを特徴とす る、中枢性疾患の解析方法。 [8] A method for analyzing a central disease, comprising using the mouse according to claim 7 or a nerve cell obtained from the mouse.
[9] 請求項 7に記載のマウス又は該マウスから得られる神経細胞に試験化合物を投与又 は添加することを特徴とする、中枢性疾患の治療薬のスクリーニング方法。 [9] A screening method for a therapeutic drug for a central disease, which comprises administering or adding a test compound to the mouse according to claim 7 or a nerve cell obtained from the mouse.
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