JP2001321017A - mu3B GENE-DEFECTIVE NON-HUMAN ANIMAL - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a μ3B knockout mouse, and to provide a method for screening a tonoclonic convulsion inhibitor and an anti-epileptic agent therewith. SOLUTION: This μ3B gene-defective mouse prepared by forming a targeting vector having an about 5 kb genome DNA fragment containing the exon 1 of a mouse μ3B gene and its 5'-flanking region, a 240 bp GFP gene connected to the translation-initiating codon of the exon 1, NEOR, the 3'-flanking region of the exon 1 of the μ3B gene, and HSV-tk gene, transducing an ES cell strain with the formed targeting vector by an electroporation method, introducing a mutation into one side of the μ3B gene by a homologous recombination method, and then transplanting the obtained ES cell clone into the uterus of a mouse. The μ3B knockout mouse responding to the change of a body position and sound stimulation to exhibit tonoclonic convulsion or epileptic syndrome is used for the screening.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a non-human animal in which the function of the μ3B gene is deficient on a chromosome, and a method for screening for a tonic-clonic seizure inhibitor or an antiepileptic agent using the same.

[0002]

2. Description of the Related Art In order for cells to function normally, individual constituent proteins must be correctly transported to their final destination, intracellular organelles, which is no exception in nerve cells. Therefore, studies on intracellular protein transport are considered important in elucidating the mechanism of expression of various cranial nerve functions and understanding the pathology of neurological diseases. Secretory proteins and membrane proteins newly synthesized in the cell are transported to the trans-Golgi network (TGN) via the endoplasmic reticulum and the Golgi, where the proteins reaching the TGN are converted there into final proteins such as the plasma membrane, endosomes and lysosomes. Assigned to destination. Transport from TGN to the plasma membrane is a constant and large-volume, default signal pathway that does not require a signal, whereas transport to endosomes and lysosomes is performed selectively over this large flow. Requires a signal.

[0003] It is believed that the transport of proteins between organelles is carried out through transport vesicles, and that plasma membrane and T
On the cytoplasmic side of GN, there is a region covered with clathrin and adapter protein (AP) complex, from which clathrin-coated vesicles, one of the transport vesicles, sprout. The AP complex is composed of four subunits,
One μ chain binds to a tyrosine transport signal present on the cytoplasmic side of the membrane protein, thereby selectively transporting a membrane protein having a signal. The recently identified AP-3 complex includes subunits β3A and μ3A expressed systemically and subunits β3B and μ3B expressed specifically in brain nerve cells (J. Cell Biol. 1).
45: 923-926, 1999), A containing these β3B and μ3B
In vitro experiments suggest that P-3B may be involved in some important higher nervous functions (Cell 93:42).
3-432, 1998), but the details were not clear.

[0004]

The problem to be solved by the present invention is that of μ3
Non-human animals such as B knockout mice lacking the μ3B gene and exhibiting involuntary tonic-clonic seizures and epileptic seizures in response to repositioning and sound stimuli, tonic-clonic seizure inhibitors using the same, An object of the present invention is to provide a method for screening for epilepsy.

[0005]

Means for Solving the Problems The present inventors have proposed AP-3.
In order to analyze the function of B at the individual level, a subunit, μ3B knockout mouse, was prepared and its properties were examined in various ways. The mechanism is not clear. The present inventor has found that the patient exhibits tonic tonic-clonic convulsions and epileptic seizure symptoms in response to the stimulus, thereby completing the present invention.

That is, the present invention relates to a non-human animal characterized in that the μ3B gene function is deficient on the chromosome (claim 1), and to the ability to express a marker substance gene. The non-human animal according to claim 1 or 2, wherein the drug resistance gene used in the screening of the homologous recombinant has been removed (claim 3), A gene-deficient non-human animal characterized by exhibiting tonic-clonic convulsions or epileptic symptoms in response to repositioning (claim 4), or exhibiting tonic-clonic convulsions or epileptic symptoms in response to sound stimulation The gene-deficient non-human animal according to claim 4 or claim 5, wherein the gene deficiency is a defect in μ3B gene function (claim 6).
The non-human animal according to any one of claims 1 to 6, wherein the non-human animal is a rodent.
And a non-human animal according to claim 7, wherein the rodent is a mouse.

[0007] The present invention also provides a method of administering a test substance to a non-human animal according to any one of claims 1 to 8, and analyzing and evaluating the degree of tonic-clonic convulsions or epileptic symptoms in the non-human animal. A method for screening a tonic-clonic seizure inhibitor or an antiepileptic agent, which is characterized in (claim 9), and claim 1
A test substance is administered to the non-human animal and wild-type non-human animal according to any one of Items 1 to 8, and the degree of tonic-clonic convulsions or epileptic symptoms in the non-human animal and the wild-type non-human animal is analyzed and compared. A method for screening for a tonic-clonic seizure inhibitor or an antiepileptic agent, characterized in that it is performed (claim 10)
And contacting a brain nerve cell obtained from the non-human animal according to any one of claims 1 to 8 with a test substance in vitro;
A method for screening an ankylosing clonic seizure inhibitor or an antiepileptic agent, characterized by analyzing and evaluating the degree of selective protein transport ability in the secretory system and / or endocytosis in the brain nerve cells (claim 11), Claim 1
9. A test substance is brought into contact with a brain nerve cell obtained from the non-human animal or wild-type non-human animal according to any one of claims 8 to 8 in vitro, and the brain nerve cell has a selective protein transport ability in a secretory system and / or endocytosis. A method for screening for an ankylosing clonic convulsion inhibitor or an antiepileptic agent, characterized by analyzing and comparing the degree of the disease (Claim 12)
Or the wild-type non-human animal is a wild-type non-human animal of the same litter as a non-human animal deficient in μ3B gene function, the tonic-clonic seizure suppression according to any one of claims 9 to 12, A method for screening an agent or an antiepileptic agent (Claim 13), a tonic-clonic seizure inhibitor or an antiepileptic agent obtained by the screening method according to any one of Claims 9 to 13 (Claim 14), SEQ ID NO: 1 And a mouse μ3B cDNA having the nucleotide sequence represented by

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as a non-human animal in which μ3B gene function is deficient on a chromosome, the endogenous gene of the non-human animal encoding μ3B is inactivated by disruption, deletion, substitution, etc. There is no particular limitation as long as the non-human animal has lost the function of expressing the gene, but the non-human animal capable of expressing the fluorescent marker gene and the drug resistance gene used in the screening of the homologous recombinant may be used. Non-human animals that have been removed are preferred. In the present invention, the gene-deficient non-human animal includes endogenous non-human animals such as the gene constituting the nervous system-specific AP complex subunit such as the μ3B gene, which are artificially impaired due to disruption, deletion, or substitution. It is not particularly limited as long as it is a non-human animal that is activated and exhibits tonic-clonic convulsions or epileptic symptoms in response to repositioning or sound stimulation.

The wild-type non-human animal in the present invention means an animal of the same species as the above-mentioned non-human animal deficient in the μ3B gene function. Above all, a litter is preferable. And, as a non-human animal deficient in μ3B gene function,
Those born according to Mendel's law are preferable in that a wild type that is the same as the μ3B-deficient type can be obtained, and an accurate comparative experiment can be performed using these types. Specific examples of these non-human animals include rodents such as mice and rats, but are not limited thereto. Hereinafter, a method for preparing the non-human animal, which is a mouse, that is, a case where the non-human animal whose μ3B gene function is deficient on the chromosome is a μ3B knockout mouse, will be described as an example.

As a method for producing a μ3B knockout mouse, any method can be used as long as it can produce a knockout mouse that has lost the function of expressing μ3B. For example, a mouse genomic DNA library is screened using rat μ3B cDNA as a probe, the gene of μ3B genomic DNA is isolated, and the exon and its surrounding introns are replaced with a selectable marker gene to produce a targeting vector. Then, the prepared targeting vector is introduced into ES cells by electroporation or the like, and an ES cell clone in which homologous recombination has occurred is selected. It is preferable to confirm whether or not the selected ES cell is the desired recombinant by Southern blotting or the like. A germ-line chimeric mouse is prepared using this ES cell clone, and heterozygous mice (F1: first-generation hybrid) obtained by crossing with a wild-type mouse are crossed with each other to produce a mouse according to Mendel's law. Μ3B knockout mouse and littermate wild type mouse can be produced.

[0011] Examples of the selective marker gene in the target vector, a neomycin resistance (NEO ^R) gene, hygromycin B gene and various drug-resistant gene, GFP (Green Fluorescence Protein) to understand the expression and location of μ3B gene in the mouse body gene , Β-galactosidase gene and other marker substances by fluorescence or coloring, the herpes simplex virus thymidine kinase (HSV-tk) gene for eliminating random recombinants that are not at predetermined positions, and the diphtheria toxin A fragment gene. be able to.
Moreover, knockout mice produced using the NEO ^R gene, such as lox P sequences are added on both sides, by crossing with transgenic mice expressing oocyte specifically cre gene, NEO ^R gene genome A knockout mouse removed from above can be produced. Furthermore, the knockout mouse thus prepared was
By mating with a transgenic mouse into which the large T antigen gene of V40 or the like has been introduced, a knockout mouse from which a μ3B gene-deficient immortalized cell line can be obtained can also be prepared.

[0012] The method of screening for an ankylosing tonic-clonic seizure inhibitor or an antiepileptic agent according to the present invention includes a method for responding to a non-human animal such as a μ3B knockout mouse whose μ3B gene function is deficient on a chromosome or to a change of body position or sound stimulation. A test substance is administered to a non-human animal exhibiting tonic-clonic convulsions or epileptic symptoms, and any screening method for analyzing and evaluating the degree of tonic-clonic convulsions or epileptic symptoms in the non-human animal or the non-human animal is particularly limited. However, in the analysis and evaluation, it is preferable to compare and evaluate with the analysis result of a wild-type non-human animal as a control. Specific examples of the administration method include oral administration and intravenous injection. Analysis of the degree of tonic-clonic convulsions or epileptic symptoms can be performed by visually observing the state of convulsions or seizures in the test non-human animal or the test wild-type non-human animal as a control, or by analyzing these non-human animals. This can be done by measuring the brain waves of the subject.

[0013] Further, other screening methods of the tonic-clonic seizure inhibitor or the antiepileptic agent of the present invention include non-human animals such as the above-mentioned μ3B knockout mouse whose μ3B gene function is deficient on the chromosome; Hippocampus of a non-human animal exhibiting tonic-clonic convulsions or epileptic symptoms in response to sound stimulation, cerebral cortex, cerebellum, cerebral nerve cells obtained by conventional methods isolated from olfactory bulb and the like, and a test substance are contacted in vitro, The screening method is not particularly limited as long as it is a screening method for analyzing and evaluating the degree of selective protein transport ability in the secretory system and / or endocytosis in the brain nerve cells. Is preferred. In the analysis and evaluation, it is preferable to compare and evaluate the results with the analysis results on brain neurons of a wild-type non-human animal as a control. The above-mentioned in vitro contact can be usually performed under the conditions of liquid phase culture of brain nerve cells and the like. Further, the analysis of the degree of the selective protein transport ability in the secretory system and / or endocytosis in the brain nerve cells can be performed by electrophysiologically measuring the release amount of a neurotransmitter in the brain nerve cells. .

The tonic-clonic seizure inhibitor or antiepileptic agent obtained by the above screening method is not only important for elucidating the mechanism of onset of tonic clonic seizures and epilepsy, but also for the onset of tonic clonic seizures and epilepsy. The potential for use as a therapeutic, symptom-improving, or prophylactic agent for patients who have been affected can be expected.

Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples. [Preparation of mouse μ3B gene] Mouse isolated from mouse brain cDNA library (Stratagene) and mouse genomic DNA library (Stratagene) using rat μ3B gene cDNA (provided by Dr. R. Scheller of Stanford University, USA) as a probe μ3B cDNA and mouse μ3B genome D
NA was used to generate the following μ3B knockout mice. SEQ ID No. 1 in the sequence listing shows the nucleotide sequence of mouse cDNA, and SEQ ID No. 2 shows its amino acid sequence.

[Construction of Targeting Vector] A sense primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a sequence of SEQ ID NO: 4 from the translation initiation codon of exon 1 of the mouse μ3B gene to the Eco RI site at about 500 bases upstream. Amplified by PCR using an antisense primer consisting of the nucleotide sequence represented by Next, GFP
PCR using a gene (Clontech) with a sense primer consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an antisense primer consisting of the nucleotide sequence shown in SEQ ID NO: 6
Amplified by The 3 'side of the antisense primer consisting of the nucleotide sequence represented by SEQ ID NO: 4 and SEQ ID NO: 5
Is complementary to the 5 'side of the sense primer consisting of the nucleotide sequence represented by the above formula. Therefore, using the two amplification products (DNA fragments) as templates, the sense primer consisting of the nucleotide sequence represented by the above SEQ ID NO: 3 Primer and SEQ ID NO: 6
Amplification by PCR using an antisense primer consisting of the nucleotide sequence represented by
A fusion DNA fragment having a reading frame with P was obtained.

PBluescript from which the Sal I site has been artificially removed
The fusion DNA fragment was inserted into the Eco RI-Hind III site of II SK (Stratagene). Of the resulting construct
About 3 kb Eco RI 5 'upstream of exon 1 at Eco RI site
The fragment was inserted. Next, pl2-neo (provided by Dr. H. Gu of the United States Health Service) added S site to the Sph I and Hind III sites located 3 'to the GFP sequence in this construct.
I was cut out with ph I and Hind III, and inserted the lox P-NEO ^R -lox P gene fragment. The thus obtained construct lox P-NEO ^R -lox P gene fragment downstream of Sal I and Cl
A targeting vector by cutting at the aI site and simultaneously inserting two DNA fragments prepared as follows, an EcoRI fragment of about 1.5 kb downstream of exon 1 and the HSV-tk gene at the site. Was built. The approximately 1.5 kb Eco RI fragment downstream of exon 1 was subcloned into Eco RI of pBluescript II SK and
Sal I-Sp by cutting with Sal I and Spe I on both sides of co RI
Prepared as eI fragment. The HSV-tk gene is
plC19R-MC1-TK (provided by Dr. K. Thomas, University of Utah)
The HSV-tk gene was cut out with Eco RI and Hind III, and pBl
After inserting the EcoRI and HindIII sites of uescript II SK, and further inserting the EcoRI fragment containing the MC1 promoter region of pMC1 neo (Stratagen) into the same EcoRI site, the entire insert obtained was Spe I-Cla I Prepared as fragments.

[Screening of homologous recombinant] The targeting vector was introduced into the ES cell line E14 by electroporation,
The S clone was selected by G418 and GANC (Ganciclovir) and screened by Southern blotting using two probes (L, S) to obtain an ES cell clone in which a mutation was introduced into one of the μ3B genes by homologous recombination. Was. The probe L shown in FIG.
An EcoRI-SspI fragment of about 500 bp of B genomic DNA was used. As the probe S, a PCR amplification product obtained using a sense primer having the nucleotide sequence of SEQ ID NO: 7 and an antisense primer having the nucleotide sequence of SEQ ID NO: 8 using μ3B genomic DNA as a template was used. FIG. 2 shows the results of the Southern blot.

[Preparation of knockout mouse]
Female BD in which S cell clone was microinjected into blastocysts of C57BL / 6 mice and blastocysts after treatment were placed in pseudopregnancy
Chimeric mice were obtained by transplantation into the uterus of F1 mice. This chimeric mouse was bred to a C57BL / 6 mouse to obtain a mouse heterozygous for the μ3B gene mutation,
By crossing them, a μ3B-deficient mouse having a homozygous μ3B gene mutation was obtained. The absence of μ3B expression in the μ3B knockout mouse was confirmed by the fact that the sense primer consisting of the nucleotide sequence represented by SEQ ID NO: 9 and the SEQ ID NO: 1
It was confirmed by RT-PCR using an antisense primer consisting of the base sequence represented by 0. Also, RT-P
As a control actin (actin) primer for CR, a sense primer consisting of the base sequence of SEQ ID NO: 11 and an antisense primer consisting of the base sequence of SEQ ID NO: 12 were used. The results are shown in FIG. And μ3B knockout mice obtained by crossing the transgenic mice (donor from Junichi Miyazaki of Osaka University Ph.D.) expressing oocyte specifically cre gene, a knockout mouse NEO ^R gene has been removed from the genome Established. By analyzing the expression of GFP in the brain of this knockout mouse, the hippocampus, cerebral cortex, cerebellum,
Expression was confirmed in a specific cell group in the olfactory bulb and the like.

[0020]

The non-human animal deficient in the .mu.3B gene such as the .mu.3B knockout mouse of the present invention exhibits involuntary tonic-clonic convulsions or epileptic symptoms in response to repositioning or sound stimulation. The use of gene-deficient non-human animals can be expected to elucidate the mechanism of onset of tonic-clonic convulsions and epilepsy. In addition, the tonic-clonic seizure inhibitor or antiepileptic agent obtained by the screening method using the μ3B gene-deficient non-human animal of the present invention is a therapeutic, symptom-ameliorating or prophylactic agent for a patient who has developed tonic-clonic seizure or epilepsy It can be expected that it can be used as

[0021]

[Sequence List] SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION <120> mu3B gene-deficient non-human animals <130> 12-048 <140> <141> <160> 12 <170> PatentIn Ver. 2.1 <210 > 1 <211> 1257 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (1257) <400> 1 atg att cac agc ctt ttc ttg att aac tct tcc gga gat att ttc ctg 48 Met Ile His Ser Leu Phe Leu Ile Asn Ser Ser Gly Asp Ile Phe Leu 1 5 10 15 gag aaa cac tgg aaa agt gtg gtc agc cgc tct gtg tgt gat tac ttc 96 Glu Lys His Trp Lys Ser Val Val Ser Arg Ser Val Cys Asp Tyr Phe 20 25 30 ttc gag gcc caa gag aga gct aca gag gca gaa aat gtg cct ccg gtc 144 Phe Glu Ala Gln Glu Arg Ala Thr Glu Ala Glu Asn Val Pro Pro Val 35 40 45 atc ccc acc ccc cac cac tac ctc ctg agc gtt tac cgg cac aag atc 192 Ile Pro Thr Pro His His Tyr Leu Leu Ser Val Tyr Arg His Lys Ile 50 55 60 ttc ttt gtg gct gta atc cag aca gag gtc ccg cct ctg ttt gtc att 240 Phe Phe Val Ala Val Ile Gln Thr Glu Val Pro Pro Leu Phe Val Ile 65 70 75 80 gaa ttc ctt cac cgc g ta gtg gat aca ttt cag gac tat ttt ggg gtc 288 Glu Phe Leu His Arg Val Val Asp Thr Phe Gln Asp Tyr Phe Gly Val 85 90 95 tgt tcc gag cca gtg atc aaa gac aat gtg gtg gtg gtg tac gag gtg 336s Ser Glu Pro Val Ile Lys Asp Asn Val Val Val Val Tyr Glu Val 100 105 110 ttg gaa gag atg ctg gac aat ggg ttc ccc ttg gct acg gag tcg aac 384 Leu Glu Glu Met Leu Asp Asn Gly Phe Pro Leu Ala Thr Glu Ser Asn 115 120 125 atc ctc aag gag ctg ata aag cct ccc acc att ctc cgg aca gta gtc 432 Ile Leu Lys Glu Leu Ile Lys Pro Pro Thr Ile Leu Arg Thr Val Val 130 135 140 aac acc atc aca gga agc acc aat gtg ggg gac cag ctt cca act ggg 480 Asn Thr Ile Thr Gly Ser Thr Asn Val Gly Asp Gln Leu Pro Thr Gly 145 150 155 160 cag ctg tcg gtg gtg cct tgg cga cgg aca ggg gtg aaa tat acc aac 528 Gln Leu Ser Val Val Pro Trp Arg Arg Thr Gly Val Lys Tyr Thr Asn 165 170 175 aac gag gcc tat ttc gat gtg gtg gaa gag ata gac gcc atc att gac 576 Asn Glu Ala Tyr Phe Asp Val Val Glu Glu Ile Asp Ala Ile Ile Asp 180 185 190 aag tca ggt tcc aca gtc acc gct gag atc caa ggg gtg atc gat gcc 624 Lys Ser Gly Ser Thr Val Thr Ala Glu Ile Gln Gly Val Ile Asp Ala 195 200 205 tgt gtt aag ctg acc ggg atg ccg gat ctc act ctg tcc ttc atg aac 672 Cys Val Lys Leu Thr Gly Met Pro Asp Leu Thr Leu Ser Phe Met Asn 210 215 220 ccc agg ttg ctg gac gat gtt agc ttc cac ccc tgt gtc cgt ttc aag 720 Pro Arg Leu Leu Asp Asp Val Ser Phe His Pro Cys Val Arg Phe Lys 225 230 235 240 cgc tgg gaa tct gag cgc atc ctc tcc ttc atc cct cct gac ggc aac 768 Arg Trp Glu Ser Glu Arg Ile Leu Ser Phe Ile Pro Pro Asp Gly Asn 245 250 255 ttc cgc ctg cc gc tc agt gca cag aac ctc gtt gcc att 816 Phe Arg Leu Leu Ala Tyr His Val Ser Ala Gln Asn Leu Val Ala Ile 260 265 270 cca gtc tat gt gtc aaa cat agc atc agc ttc cgg gac agc agt tca ctt 864 Pro Val Tyr Vals His Ser Ile Ser Phe Arg Asp Ser Ser Ser Leu 275 280 285 gga cgc ttt gaa atc acg gtg gga ccc aag cag acc atg ggg aag acc 912 Gly Arg Phe Glu Ile Thr Val Gly Pro Lys Gln Thr Met Gly Lys Thr 290 295 300ata gaa ggt gtg att gtc acc agc cag atg ccc aaa gga atc ctg aat 960 Ile Glu Gly Val Ile Val Thr Ser Gln Met Pro Lys Gly Ile Leu Asn 305 310 315 320 ata agc ctc act cca tct cag ggg aca cac acg ttt g ccc gtg aca 1008 Ile Ser Leu Thr Pro Ser Gln Gly Thr His Thr Phe Asp Pro Val Thr 325 330 335 aaa atg ctg tct tgg gat gta gga aaa cta aac caa caa aag ctg cca 1056 Lys Met Leu Ser Trp Asp Val Gly Lys Leu Asn Gln Gln Lys Leu Pro 340 345 350 agt ttg aag ggg acg atg ggt ctc cag gtc gga gct tct aaa cca gat 1104 Ser Leu Lys Gly Thr Met Gly Leu Gln Val Gly Ala Ser Lys Pro Asp 355 360 365 365 gag aac cct acg att aac ctg cag ttt aag atc cag cag ctg gcc att 1152 Glu Asn Pro Thr Ile Asn Leu Gln Phe Lys Ile Gln Gln Leu Ala Ile 370 375 380 tct gga ctc aaa gtg aac cgt ctg gat atg tac ggg gag aag tac aag 1200 Leu Lys Val Asn Arg Leu Asp Met Tyr Gly Glu Lys Tyr Lys 385 390 395 400 400 ccc ttt aaa ggc atc aaa tac atg acc aaa gcc ggc aag ttc caa gtc 1248 Pro Phe Lys Gly Ile Lys Tyr Met Thr Lys Ala Gly Lys Ph e Gln Val 405 410 415 cga acc tga 1257 Arg Thr <210> 2 <211> 418 <212> PRT <213> Mus musculus <400> 2 Met Ile His Ser Leu Phe Leu Ile Asn Ser Ser Gly Asp Ile Phe Leu 1 5 10 15 Glu Lys His Trp Lys Ser Val Val Ser Arg Ser Val Cys Asp Tyr Phe 20 25 30 Phe Glu Ala Gln Glu Arg Ala Thr Glu Ala Glu Asn Val Pro Pro Val 35 40 45 Ile Pro Thr Pro His His Tyr Leu Leu Ser Val Tyr Arg His Lys Ile 50 55 60 Phe Phe Val Ala Val Ile Gln Thr Glu Val Pro Pro Leu Phe Val Ile 65 70 75 80 Glu Phe Leu His Arg Val Val Asp Thr Phe Gln Asp Tyr Phe Gly Val 85 90 95 Cys Ser Glu Pro Val Ile Lys Asp Asn Val Val Val Val Tyr Glu Val 100 105 110 Leu Glu Glu Met Leu Asp Asn Gly Phe Pro Leu Ala Thr Glu Ser Asn 115 120 125 Ile Leu Lys Glu Leu Ile Lys Pro Pro Thr Ile Leu Arg Thr Val Val 130 135 140 Asn Thr Ile Thr Gly Ser Thr Asn Val Gly Asp Gln Leu Pro Thr Gly 145 150 155 160 Gln Leu Ser Val Val Pro Trp Arg Arg Thr Gly Val Lys Tyr Thr Asn 165 170 175 Asn Glu Ala Tyr Phe Asp Val Val Glu Glu Ile Asp Ala Ile Ile Asp 180 185 190 Lys Ser Gly Ser Thr Val Thr Ala Glu Ile Gln Gly Val Ile Asp Ala 195 200 205 Cys Val Lys Leu Thr Gly Met Pro Asp Leu Thr Leu Ser Phe Met Asn 210 215 220 Pro Arg Leu Leu Asp Asp Val Ser Phe His Pro Cys Val Arg Phe Lys 225 230 235 240 Arg Trp Glu Ser Glu Arg Ile Leu Ser Phe Ile Pro Pro Asp Gly Asn 245 250 255 Phe Arg Leu Leu Ala Tyr His Val Ser Ala Gln Asn Leu Val Ala Ile 260 265 270 Pro Val Tyr Val Lys His Ser Ile Ser Phe Arg Asp Ser Ser Ser Leu 275 280 285 Gly Arg Phe Glu Ile Thr Val Gly Pro Lys Gln Thr Met Gly Lys Thr 290 295 300 Ile Glu Glu Val Ile Val Thr Ser Gln Met Pro Lys Gly Ile Leu Asn 305 310 315 320 Ile Ser Leu Thr Pro Ser Gln Gly Thr His Thr Phe Asp Pro Val Thr 325 330 335 Lys Met Leu Ser Trp Asp Val Gly Lys Leu Asn Gln Gln Lys Leu Pro 340 345 350 Ser Leu Lys Gly Thr Met Gly Leu Gln Val Gly Ala Ser Lys Pro Asp 355 360 365 Glu Asn Pro Thr Ile Asn Leu Gln Phe Lys Ile Gln Gln Leu Ala Ile 370 375 380 Ser Gly Leu Lys Val Asn Arg Leu Asp Met Tyr Gly Glu Lys Tyr Lys 385 390 395 400 Pro Phe Lys Gly Ile Lys Tyr Met Thr Lys Ala Gly Lys Phe Gln Val 405 410 415 Arg Thr <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 47B3K-3 '(Sense) <400> 3 gacaaacaag ttggctgcta g 21 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 47B3K-3' (Antisense) <400> 4 ctcgcccttg ctcaccatgg tggagaagag gccggt 36 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: GFP Poly A-Sense <400> 5 accggcctct ccaccatggt gagcaagggc gag 33 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: GFP Poly A-Antisense <400> 6 cccaagcttg gggcatgcta agatacattg atgagtttgg 40 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: p47B-1.5kb-3 '(Sense) <400> 7 gagcaagttt gttctgtcca c 21 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificia l Sequence: p47B-0.5kb-Antisense1 <400> 8 tatggggtat cgtacttaga aatcacagca gg 32 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mu3B primer (Sense ) <400> 9 caccggcctc ttctccacca tg 22 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mu3B primer (Antisense) <400> 10 gatggcgtct atctcttcca c 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: actin primer (Sense) <400> 11 acccacactg tgcccatcta 20 <210> 12 <211> 20 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: actin primer (Antisense) <400> 12 tcatggatgc cacaggattc 20

[Brief description of the drawings]

FIG. 1 is a diagram showing a genetic map of a μ3B knockout mouse and a wild-type mouse of the present invention.

FIG. 2 shows the results of Southern blot in screening for homologous recombinants.

FIG. 3 shows that μ3B knockout mice of the present invention do not express μ3B.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) G01N 33/15 C12N 5/00 B 33/50 15/00 ZNAA F term (reference) 2G045 AA29 AA40 CB17 GC30 4B024 AA11 AA20 BA80 CA04 DA02 EA04 GA11 HA20 4B065 AA91X AA91Y AB01 BA02 CA46 4C084 AA16 NA14 ZA022 ZA062

Claims (15)

[Claims] 1. A non-human animal characterized in that the μ3B gene function is deficient on a chromosome. 2. The non-human animal according to claim 1, wherein the non-human animal has an ability to express a marker substance gene. 3. The non-human animal according to claim 1, wherein the drug resistance gene used in the screening of the homologous recombinant has been removed. 4. A gene-deficient non-human animal characterized by exhibiting tonic-clonic convulsions or epileptic symptoms in response to repositioning. 5. A gene-deficient non-human animal characterized by exhibiting tonic-clonic convulsions or epileptic symptoms in response to a sound stimulus. 6. The gene-deficient non-human animal according to claim 4, wherein the gene deficiency is a deficiency in μ3B gene function. 7. The non-human animal according to claim 1, wherein the non-human animal is a rodent. 8. The non-human animal according to claim 7, wherein the rodent is a mouse. 9. A test substance is administered to the non-human animal according to any one of claims 1 to 8, and the degree of tonic-clonic convulsions or epileptic symptoms in the non-human animal is analyzed and evaluated. A method for screening an ankylosing clonic seizure inhibitor or an antiepileptic agent. 10. A tonic-clonic convulsion or epilepsy in a non-human animal and a wild-type non-human animal by administering a test substance to the non-human animal or wild-type non-human animal according to any one of claims 1 to 8. A method for screening an ankylosing clonic convulsion inhibitor or an antiepileptic agent, comprising analyzing and comparing the degree of symptoms. 11. A test substance is brought into contact with a brain nerve cell obtained from the non-human animal according to any one of claims 1 to 8 in vitro, and a selective protein in the secretory system and / or endocytosis in the brain nerve cell is obtained. A screening method for an ankylosing clonic convulsion inhibitor or an antiepileptic agent, which comprises analyzing and evaluating the degree of transport ability. 12. A secretory system and / or an endothelium in a brain nerve cell obtained by contacting a brain nerve cell obtained from the non-human animal or wild-type non-human animal according to any one of claims 1 to 8 with a test substance in vitro. A method for screening a tonic-clonic seizure inhibitor or an antiepileptic agent, comprising analyzing and comparing the degree of selective protein transport ability in cytosis. 13. The tonic as claimed in any one of claims 9 to 12, wherein the wild-type non-human animal is a wild-type non-human animal of the same litter as a non-human animal deficient in μ3B gene function. Screening method for anticonvulsant or antiepileptic agent. A tonic-clonic seizure inhibitor or an antiepileptic agent obtained by the screening method according to any one of claims 9 to 13. 15. A mouse μ3B cDNA comprising the nucleotide sequence represented by SEQ ID NO: 1.