JP2003158954A - New nonhuman animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nonhuman animal with an expression level of brain immunoglobulin molecule with tyrosine activation motifs (BIT) gene knockouted or reduced from that of a wild type. <P>SOLUTION: This nonhuman animal is prepared by constructing a targeting vector for making a region containing an initiation codon of a first exon of the BIT gene deficient, transducing the resultant construct into an ES cell, producing a homologous recombinant and mating the resultant recombinant with a chimeric mouse prepared from the ES cell. The nonhuman animal has the expression level of the BIT gene knockouted or reduced from that of the wild type. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a non-human animal in which the expression level of the BIT gene disappears or is reduced as compared with the wild type, or a progeny thereof. Specifically, it relates to a non-human animal in which the BIT gene is disrupted at the BIT locus on a homologous chromosome, and its use.

[0002]

2. Description of the Related Art BIT protein (BIT; Brain i)
mmunoglobulin-like molecu
le with tyrosine-based ac
Tivation motifs (TAMs) are membrane-bound glycoproteins that are highly expressed in the brain, and are involved in cell-cell recognition and adhesion in the extracellular part (Sano S., e.
t al. , FEBS Lett. , 411, 327-
334 (1997)), which is involved in signal transduction in the intracellular part (Ohnishi H., et al., J. Bio.
l. Chem. , 271, 25569-25574 (1
996)) has been elucidated.

For example, the BIT protein is known to be involved in connection and recognition between nerve cells, signal transmission of nerve cells, and the like, and is also known to play an important role in survival, differentiation and plasticity of nerve cells. NGF (never g
lower factor), BDNF (brain-
Derived neurotrophic fact
or), NT-3 (neurotrophin-3) and other neurotrophins have been involved in signal transduction (Ohnishi H., et al.,
J. Neurochem. , 72, 1402-1408
(1999)). Although the mechanism of action of antidepressants is still unclear, there is a theory that the function of BDNF is important for the effects of antidepressants following activation of monoamine nerves (Alter CA, Trends.
Pharmacol. Sci. , 20, 59-61
(1999); Duman R. et al. S. et al. ，
Arch. Gen. Phychiatry, 54, 59
7-606 (1997)), it is also reported that the expression of BDNF is increased by antidepressants and that BDNF has an antidepressant-like action (Siuciak J. et al.
A. , Pharmacol. Biochem. Beha
v. 56, 131-137 (1997)). Since the BIT protein is involved in the action of BDNF, it is considered to be a molecule useful for elucidating the mechanism of action of antidepressants and developing antidepressants.

Further, the BIT protein is widely expressed in various in vivo tissues, although the amount thereof is smaller than that in the brain (S
ano S. , Et al. , Biochem. J. ，
334, 667-675 (1999)). Especially, the importance of physiological roles in the nervous system and the immune system has been paid attention to.
HPS-1 (Fujioka Y., et al., M
ol. Cell. Biol. , 16,6887-689
9 (1996)), SIRP (Kharitennk)
ov A. , Et al. , Nature, 386, 1
81-186 (1997)), P84 (Comu
S. , Et al. J. Neurosci. , 17,
8702-8710 (1997)), MFR (Sagi
nario C. , Et al. , Mol. Cell.
Biol. , 18, 6213-6223 (1998)).
Has been widely studied under the name.

The function of the BIT protein has been elucidated also in the immune system. Among them, the CD47 molecule, which is a membrane-bound protein, has been reported as an extracellular ligand of the BIT protein (Jiang P., et. al., J.
Biol. Chem. , 274, 559-562 (19
99)). In the immune system, the BIT protein is present in macrophages, dendritic cells, etc., and when the CD47 molecule on erythrocytes is not recognized by the BIT protein on spleen macrophages, erythrocytes are phagocytosed by macrophages (Olde.
nburg P. A. , Et al. , Science
e, 288, 2051-2054 (2000)). In addition, when macrophages fuse and change into multinucleated cells (osteoclasts, giant cells), CD
47 has also been reported to be involved (Han X., e
t al. J. Biol. Chem. , 275, 37
984-37992 (2000)).

Other BIT proteins include insulin, E
GF (epidermal growth facto)
r), PDGF (platelet-delivered)
It has also been shown to be involved in signal transduction using many tyrosine phosphorylations by receptors such as growth factor (Fujioka Y., et a).
l. , Mol. Cell. Biol. , 16,6887
-6899 (1996); Kharitonenkov
A. , Et al. , Nature, 386, 181
-186 (1997)). It also plays an important role in integrin signaling (Tsuda).
M. , Et al. J. Biol. Chem. , 27
3, 13223-13229 (1998)).

[0007] As described above, the BIT protein is a molecule which is widely distributed in various tissues in the living body and is known to fulfill many important functions mainly in the nervous system and the immune system.
Therefore, in order to analyze the function of the BIT protein, a mouse in which only the intracellular part of the BIT protein has been artificially deleted has been prepared (Inagaki K., et a.
l. , EMBO J .; , 19, 6721-6731 (2
000)). However, in this mouse, only part of the expression of the BIT gene has disappeared or decreased, and there is no report of behavioral abnormalities in the created mouse. That is, B
No animal in which full-length expression of the IT gene has disappeared or decreased has been obtained so far, and such an animal has been used to produce BI.
It was expected that the function of the T protein would be further analyzed, and that elucidation of cranial nervous system diseases, immune system diseases, and the like would be carried out.

[0008]

DISCLOSURE OF THE INVENTION The present invention has been made to provide a non-human animal in which the expression level of the BIT gene has disappeared or decreased compared to the wild type.

[0009]

Means for Solving the Problems As a result of intensive studies in view of the above problems, the present inventors constructed a targeting vector for deleting the region containing the start codon of the first exon of the BIT gene, and It was found that a mouse in which the expression level of BIT gene is lost or reduced can be prepared by introducing H. coli into ES cells to cause homologous recombination and mating the chimeric mice prepared by the ES cells. In addition, it was found that the produced mouse has a depression-like symptom. The present invention has been accomplished based on these findings.

That is, according to the present invention, (1) a non-human animal in which the expression level of BIT gene is reduced or decreased compared to the wild type, or a progeny thereof, and (2) the loss of expression level of BIT gene is homologous. The animal according to (1) above, which is due to the disruption of the BIT gene at two BIT loci on the chromosome, and (3) a decrease in the expression level of the BIT gene results in one BIT locus on the homologous chromosome. At B
The animal according to (1) above, which is due to the IT gene being disrupted, (4) The disruption of the BIT gene is caused by the deletion of the start codon of the gene DNA (2) or (3). (5) The animal according to (1), wherein the animal has a depression-like phenotype.
The animal according to any one of (4), (6) the animal according to any of (1) to (5) above, wherein the animal is a rodent, and (7) the rodent is a mouse. An animal according to (6) above is provided.

From another point of view, (8) above (1)
To (7), or a biological sample obtained from the animal, to which a test substance is added, and the effect of the test substance on the animal or the biological sample is detected to detect the BIT protein. A method for screening a therapeutic and / or prophylactic drug for a disease associated with methionine, (9) the method according to (8) above, wherein the disease associated with the BIT protein is depression,
0) A therapeutic and / or prophylactic agent for a disease involving a BIT protein containing the substance selected by the screening method according to (8) above as an active ingredient, (11) a substance according to the screening method according to (8) above The present invention provides a method for formulating a therapeutic and / or prophylactic agent for a disease associated with BIT protein, which comprises selecting the substance and formulating the substance.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below. In the present specification, molecular biological methods such as DNA preparation and vector preparation, protein chemical methods such as protein extraction and Western blotting, and knockout animal production operations are not particularly specified. As long as Molecular Cloning, A Lab
oratory Manual (T. Maniatis
et al. , Cold Spring Harbo
r Laboratory (1982)), New Chemistry Experiment Course (edited by the Japanese Biochemical Society; Tokyo Kagaku Dojin), Gene Targeting (Shinichi Aizawa; Yodosha (1995)), etc. be able to. 1. BIT gene expression level disappeared or low compared to wild type
Non-human animal in which the expression level of BIT gene of the present invention is reduced or decreased as compared with the wild type is a BIT in the animal.
Any gene may be used as long as the gene expression level is lost or reduced as compared with the wild type. Examples of the method for controlling the expression level of the BIT gene include, for example, B at two or one BIT loci on the homologous chromosome of the animal.
IT gene disruption method, drug administration, RNA interference method (Fire A., et al., N
ature, 391, 806-811 (1998)),
Alternatively, a method for selecting a natural mutant may be used. In addition, an animal in which the expression of the BIT gene is substantially eliminated or reduced due to mutation of other genes or the like is also included in the non-human animal of the present invention. In the present invention, the BIT gene whose expression is lost or reduced means the full length of the BIT gene, and does not include those whose expression is partially lost or reduced.

In order to prepare a non-human animal in which the expression of the BIT gene is lost due to the disruption of the BIT gene, both B of the two BIT loci on the homologous chromosome of the animal can be prepared.
A method of disrupting the IT gene is used. Further, in order to produce a non-human animal in which the expression of the BIT gene is reduced, a method of disrupting one of the BIT loci of the above animals is used. A non-human animal in which one of the BIT gene loci is disrupted is hereinafter referred to as a "heterozygous animal", and a non-human animal in which both BIT genes are disrupted is hereinafter referred to as a "homogeneous animal". . Here, the disruption of the BIT gene includes, for example, the introduction of some artificial mutation. As a specific example of the disruption of the BIT gene by artificial mutation, all of the BIT gene has D having another nucleotide sequence such as a drug resistance gene.
NA may be deleted on the genome by replacement, or BI may be deleted.
Examples thereof include a method in which a portion having the start codon of the T gene is replaced with a DNA having another nucleotide sequence on the genome to be deleted.

The BIT gene of the present invention means a DNA that provides all the genetic information about the BIT protein, specifically, a coding region translated into amino acids and an intron existing in the region on the genome. , And 5'upstream or 3'downstream of the coding region, including a non-translated region including a transcription start point and the like. This BIT gene is described, for example, in Sano S. , Et al. , Bioc
hem. J. , 334, 667-675 (1999), and includes polymerase chain reaction (PCR) and B from genomic DNA libraries and the like.
It can be obtained by hybridization using a part of the sequence of IT gene as a probe. Isolated from animals carrying the BIT gene by commonly used methods,
It can also be obtained by extracting. In the present invention, the expression of the BIT gene means the production of mRNA encoding the BIT protein and the production of BIT protein.

As the non-human animal, any animal other than human having the BIT gene can be used, but non-human mammals are preferable. Non-human mammals include, for example, cows, pigs, sheep, goats, monkeys, dogs, cats, rabbits, guinea pigs, hamsters, mice, rats, etc., among which the ontogeny and biological cycle are relatively short. Further, rodents, particularly mice, which are easy to breed are preferable. As a mouse used, for example, as a pure strain, C57BL / 6 strain, BAL
B6C3F1 as a hybrid system such as B / c system and DBA2 system
System, BDF1 system, B6D2F1 system, ICR system, etc., among which C57BL / 6 system is preferably used.

2. BIT gene expression level is higher than wild type
Production of non-human animal that has disappeared or decreased 1. Among the non-human animals whose BIT gene expression level described in 1) has disappeared or decreased compared to the wild type, those produced by disruption of the BIT gene (hereinafter, referred to as "BIT
The term “knockout animal” may be referred to) in more detail. The animal can be produced, for example, using non-human animal ES cells (embryonic stem cells) in which the BIT gene is disrupted. The BIT gene of ES cells can be destroyed by the method described above. In particular,
A targeting vector for substituting a part or all of the BIT gene with a DNA having another sequence such as a drug resistance gene on the genome is introduced into the ES cell, and homologous recombination occurs to cause BIT. This can be done by selecting cells in which the gene has been disrupted.

(1) Preparation of targeting vector The targeting vector for the BIT gene is intended to delete a part or all of the BIT gene.
When a part of the BIT gene is deleted, for example, the reading frame of the codon is displaced or the promoter or exon is inactivated, so that the BIT gene is destroyed. Deletion of a part or all of a gene by such a targeting vector is usually performed by replacement with a DNA having another sequence by homologous recombination. D to be replaced
NA (hereinafter sometimes referred to as “inserted DNA”) includes drug resistance genes such as neomycin resistance (neo) gene and hygromycin resistance gene, or lacZ (β-galactosidase gene) and cat.
A reporter gene such as (chloramphenicol acetyltransferase gene) is preferable because it can serve as a marker for homologous recombination. Furthermore, poly
Substitution with a nucleotide sequence such as A addition signal that terminates transcription of a gene can also inhibit the synthesis of complete mRNA.

As such a targeting vector, DNAs 5'upstream and 3'downstream of the portion to be deleted in the BIT gene (hereinafter referred to as "homologous region")
DNA having a structure in which the inserted DNA is sandwiched between (hereinafter, referred to as "targeting unit") may be inserted into an appropriate cloning vector as necessary. Used. Here, the homologous region may be any region as long as it is long enough to induce homologous recombination, and specifically, a region having a length of, for example, 0.5 kb or more is preferably used.
In addition, those DNA sequences have BI
It is not necessary to completely match with the T gene or the sequence in the vicinity thereof, and those having 80% or more homology are preferably used. Such a DNA fragment can be obtained from the BIT gene using the PCR method or the like. For the purpose of deleting the entire BIT gene, the targeting unit has a structure in which an appropriate inserted DNA is sandwiched between DNAs 5 ′ upstream and 3 ′ downstream of the BIT gene.
When the region having the start codon of the first exon is to be deleted, the targeting unit is B
5'upstream intron of the first exon of the IT gene and the first
Appropriate insertion DN to DNA consisting of intron sequence 5'upstream region and 3'downstream from the start codon of exon
It has a structure in which A is sandwiched.

The inserted DNA may be selected from the above-mentioned ones and used. Specifically, for example, a neo cassette "pKJ2" (Boer P. et al., 1989) in which a neomycin resistance gene and a PGKpolyA signal are linked downstream of a phosphoglycerate kinase (PGK) promoter.
H. , Et al. , Biochemical Gen
etics, 28, 299-308 (1990)) and the like can be used.

Further, in the targeting vector of the present invention, recombination has occurred by using a target gene having a different selectable marker gene inserted 5'upstream or 3'downstream of the targeting unit. Cells that have undergone random recombination that is not homologous recombination can be excluded. For example, as another marker, a gene involved in nucleic acid synthesis such as thymidine kinase gene has been inserted, so that FI added to the medium
AU (1- (2-deoxy-2-fluoro-β-
D-arabinofuranosyl) -5-ind
Ouracil), ganciclovir, and other nucleic acid synthesis inhibitors can be incorporated into cells in which random homologous recombination has occurred, and then killed and eliminated. Examples of the thymidine kinase (TK) gene include the MC1 promoter (Thomas KR and Capecc).
hi M. R. , Cell, 51, 503-512 (1
987)), and the herpes simplex virus thymidine kinase gene was ligated to the downstream of pBS.
MC1-TK cassette "F3" cloned into the EcoRI / EcoRV site of IISK (-) can be used. As a cloning vector, pBS
(Manufactured by Stratagene), pBluescriptII
(Manufactured by Stratagene), pBR322 (TAKARA
Company), pUC18, and pUC19 (manufactured by TAKARA). In addition, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, vaccinia virus or adenovirus vector, baculovirus, bovine papilloma virus, viruses from the herpesvirus group, or animal viruses such as Epstein-Barr virus. Is used.

(2) Introduction of targeting vector into non-human ES cell and selection of homologous recombinant Next, the targeting vector constructed as described above is introduced into non-human ES cell to improve efficiency. Therefore, it is preferable to introduce the targeting vector after linearizing it. The cutting point for linearization may be anywhere outside the targeting unit. Examples of the introduction method include an electroporation method (electroporation method) and a microinjection method. Among them, the electroporation method is preferable. As the non-human ES cells, those that have already been established and preserved may be used, or they may be newly established and used according to a commonly used production method known per se. As the non-human animal used for establishing non-human ES cells, the same animals as those described above can be used. For example, in the case of mouse, ES cells such as 129 line are preferably used, and among them, E14TG2a strain (Hooper
M. , Et al. , Nature, 326, 292-
295 (1987)) and the like are preferably used.

The non-human ES cells into which the targeting vector thus obtained has been introduced are cultured under conditions such that only cells in which homologous recombination has occurred can be selectively cultured. Such culture conditions may be appropriately selected according to the non-human ES cells used. For example, when a drug resistance gene is introduced, the drug used for selection is added to the medium at various concentrations, It may be determined by examining the concentration at which ES cells before the gene is introduced cannot grow. Specifically, for example, when a targeting vector in which a neomycin resistance gene and a thymidine kinase gene are inserted as the selectable marker is introduced into ES cells of 129 strain mice, for example, in a medium, 50 to 500 μm is introduced.
Culture may be performed by adding g / ml of neomycin analog such as G418 (manufactured by GIBCO, etc.) and 0.1 to 5 μM of nucleic acid synthesis inhibitor such as FIAU. The neomycin analog may be arbitrarily selected from aminoglycoside antibiotics inactivated by the neomycin resistance gene. As the medium, any commonly used medium known per se can be arbitrarily selected and used.

Then, the non-human E cultured as described above is used.
A sufficient number of colonies were picked up from S cells and re-seeded, and these were subjected to Proteinase K treatment, heat treatment, etc. to extract DNA, genotype analysis was performed, and homologous recombination occurred in the BIT gene. Non-human ES
The cells (hereinafter, sometimes referred to as "homologous recombinants") can be more reliably selected. The genotype can be analyzed by PCR using a primer consisting of a nucleotide sequence of the inserted DNA and a nucleotide sequence of a region not deleted by homologous recombination, or a nucleotide sequence of the region not deleted by the insert DNA or by homologous recombination. And a Southern hybridization method using a probe consisting of Any primer or probe can be used as long as it can be confirmed that homologous recombination has occurred at a desired position on the genome of the ES cell and the target DNA fragment has been replaced. Good. As a specific example, for example, when homologous recombination in which the region containing the start codon of the first exon is replaced with a neomycin resistance gene is performed, the primer used for analysis is a primer consisting of a partial sequence of the neomycin resistance gene, Examples include a combination of primers consisting of a partial sequence of an intron 3 ′ downstream of the first exon of BIT gene, and specifically, a sense primer having the base sequence shown in SEQ ID NO: 5 in the sequence listing and SEQ ID NO: 6 And a combination of antisense primers having the nucleotide sequence described in 1. In addition, the probe comprises a nucleotide sequence further 5 ′ upstream of the homologous region 5 ′ upstream of the first exon of the BIT gene, the first
Those consisting of a base sequence 5'from the start codon of an exon, those consisting of a partial base sequence of the neomycin resistance gene, and those consisting of a partial base sequence of an intron 3'downstream of the first exon (specifically, respectively , Probe I and probe II, which correspond to probe neo and probe III, which will be described in detail in Example 2 below), and the like.

The homologous recombinant thus obtained is
Usually, its proliferative property is good, but since it is easy to lose the ability to develop ontogeny, careful subculture is necessary. Culturing can be carried out by a commonly used method known per se, for example, LIF (1-10000 U) on an appropriate feeder cell such as STO fibroblast.
/ Ml) in the carbon dioxide incubator (preferably 5%
Carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide,
It can be cultivated by a method such as culturing at about 37 ° C in 90% air). At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin /
Treatment with 0.1-5 mM EDTA, preferably about 0.1% trypsin / 1 mM EDTA solution) is performed to form single cells and seeding on newly prepared feeder cells. As long as the obtained homologous recombinant can stably maintain the ability of ontogeny development, the E
The method and conditions suitable for the S cells may be selected. Specifically, for example, E14T of 129 strain mouse-derived ES cells is used.
When the G2a strain is used, feeder cells are unnecessary and the culture can be performed under the same conditions as the culture before selection. It is preferable to carry out the passage usually every 1-3 days, but it is desirable to observe the cells each time and to discard the cultured cells when morphologically abnormal cells are found.

(3) Preparation of chimeric animals and BIT knockout animals The homologous recombinants thus obtained are cloned, and the cells are cloned at an appropriate stage in the early stage of embryogenesis, for example, 8-cell stage non-human animal embryo or scutellum. The resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human animal. Alternatively, a chimeric embryo may be produced using an agglutination method in which it is co-cultured with an early embryo, and this may be transplanted. The produced animal is a chimeric individual composed of both cells having a normal BIT gene and cells having a disrupted BIT gene (hereinafter, this may be referred to as "chimeric animal"). From a population of individuals obtained by mating such a chimeric individual with a normal individual (hereinafter, this may be referred to as “F1”), the BIT gene at any one of the BIT loci on the homologous chromosome Genotype destroyed (hereinafter,
A heterozygous animal can be obtained by selecting an individual having this (sometimes referred to as "heterozygous"). The heterozygous animal thus obtained is a non-human animal in which the expression level of BIT gene is lower than that of the wild-type animal. Usually, the individuals are bred and the It is possible to obtain a homozygous animal having a genotype in which the BIT gene is disrupted in the above two BIT loci (hereinafter, this may be referred to as “homotype”).
Such a homozygous animal is a non-human animal in which the BIT gene expression level is lost.

The heterozygous or homozygous animal can be selected by, for example, analyzing the genotype by Southern blotting or PCR using DNA extracted from the tail of the animal as a template. Any primer may be used as a primer used in the analysis by the PCR method as long as the genotype can be distinguished from the wild type, the hetero type, and the homo type. For example,
Examples include a method of simultaneously using a primer consisting of a partial base sequence of an inserted DNA, a primer consisting of a partial base sequence of a region deleted by homologous recombination, and a primer consisting of a partial base sequence of a region not deleted by homologous recombination.

As a specific example, when homologous recombination is performed in which the region containing the start codon of the first exon of the BIT gene is replaced with a neomycin resistance gene, a primer consisting of a partial base sequence of the neomycin resistance gene, the first exon of the BIT gene is used. PCR using the DNA extracted from F1 as a template, using a primer consisting of a partial base sequence of the region containing the start codon and an antisense primer consisting of a partial base sequence of the 3'downstream intron of the BIT gene first exon, By detecting the obtained PCR product, homotype, heterotype, or wild type can be distinguished. Specifically, for example, SEQ ID NO: in the sequence listing:
By detecting the BIT gene in which homologous recombination has occurred with the combination of the primers described in 5 and 7, and detecting the normal BIT gene with the combination of the primers described in SEQ ID NOs: 8 and 7, the non-human of the present invention can be detected. The genotype of the animal can be analyzed.

If the strain of the non-human animal from which the ES cells were obtained and the strain used for crossing are different, B
By utilizing the fact that the IT gene has a polymorphism in the nucleotide sequence portion encoding the V domain of the BIT protein, PCR is carried out using a primer capable of amplifying the nucleotide sequence portion having the polymorphism, Genotypes can be distinguished. Specifically, for example, when a 129 line mouse ES cell is transplanted into a C57BL / 6N line mouse to prepare a chimeric mouse, the primers having the sequences shown in SEQ ID NOs: 9 and 10 of the sequence listing are used. PCR using the DNA extracted from F1 as a template to obtain P
When the CR product is cleaved with the restriction enzyme KpnI, the partial nucleotide sequence encoding the V domain in the normal BIT gene derived from the C57BL / 6N system used for crossing is not cleaved, but the V domain derived from the 129 system (ES cell) The partial base sequence coding for is cleaved in two. The genotype of F1 can be identified by detecting this.

The expression of the BIT protein in the obtained heterozygous and homozygous animals can be analyzed by Western blotting or the like. Specifically, for example, a protein extract is prepared from various organs, tissues, cells, etc. of the prepared heterozygous and homozygous animals, the extract is electrophoresed, and then transferred to an appropriate membrane, The BIT protein transcribed above is converted into anti-BIT protein serum (Sano
S. , Et al. J. Biochem. , 105,
457-460 (1989)) and the like. Preparation of a protein extract, electrophoresis, transfer to a membrane, detection of a protein and the like can be carried out according to commonly known methods known per se.

(4) Passage and maintenance of heterozygous and homozygous animals The heterozygous and homozygous animals thus produced are
For animal individuals obtained by mating, it is possible to confirm that the expression level of the gene has disappeared or decreased, and perform breeding subculture in a normal breeding environment. That is, by mating a female and a male of a heterozygous animal in which the BIT gene is disrupted at one BIT locus on the homologous chromosome, B at two BIT loci on the homologous chromosome is obtained.
Homozygous and heterozygous animals in which the IT gene is disrupted can be passaged. In addition, the system can be stably maintained by mating heterozygous animals with wild-type animals. The progeny thus obtained are also included in the non-human animal of the present invention.

(5) Behavioral Analysis of BIT Knockout Animal The heterozygous and homozygous animals thus obtained can be further analyzed by behavioral analysis for the purpose of analyzing the function of the BIT protein in vivo. Any behavior analysis method may be used as long as it can be applied to the animal, such as CURRENT PROTOCOLS IN.
NEUROSCIENCE (Jhon Wiley & S
ons, Inc. ), Animal behavioral function test (biological science, published by Ikushosho, vol.45, No.5 (199
5)) and the like. Specific examples include an open field test, a rod crossing balance sensation test, a wire dangling muscle strength test, a light / dark selection test, a fear conditioning test, an electric shock sensitivity test, a Morris water maze test, and a forced swimming test. Among them, a forced swimming test, a Morris water maze test, and the like are preferably used for BIT knockout animals, but it is preferable to perform analysis by combining several methods.

The behavior analysis methods as described above are
Conditions are set so that changes in certain phenotypes such as physical ability, learning ability, or emotion can be detected. Therefore, such behavioral analysis was performed on the above-mentioned animal and wild-type animal (control), and the respective results were compared. It can be considered that the non-human animal of the present invention has some change or abnormality. Whether or not the difference between the obtained numerical values is significant can be analyzed by an appropriate index, but specifically,
For example, a statistical analysis method that is generally used, such as a t-test method, a one-way analysis of variance or a two-way analysis of variance followed by a least significant difference method, can be used. Such statistical analysis can be performed, for example, by StatView J
(Abacus Concepts, Inc.) and other statistical analysis software can be used to simplify the procedure. BIT knockout animals, for example, show significant differences from wild type in a forced swim test. Since the forced swimming test is a test in which a significant difference is detected in an animal having a cranial nerve system disease such as depression, the animal can be used as a model animal for depression and a cranial nerve system disease such as depression. It is found to be useful for screening therapeutic and / or preventive agents for

3. Treatment of diseases involving BIT protein
And / or screening for prophylactic agents The non-human animal of the present invention has a BIT gene expression level that is lost or reduced as compared with the wild type, and is therefore caused by the loss of various biological activities caused in vivo by the BIT protein. It can be used as a disease model and is useful for investigating the cause of the disease and studying the treatment method. That is, the non-human animal of the present invention can be used for screening a therapeutic and / or prophylactic drug for a disease involving the BIT protein. Such diseases include, for example, cranial nervous system diseases,
Immune system diseases and the like can be mentioned, and specifically, depression and the like can be mentioned.

It is also possible to obtain an animal in which another gene is knocked out of the non-human animal of the present invention and use it as a model animal for a disease involving a plurality of genes.
Such an animal can be obtained, for example, by mating the non-human animal of the present invention with a non-human animal in which the expression of another specific gene is lost or reduced. Specific genes include, for example, the CD47 gene known as an extracellular ligand of the BIT protein, the protein tyrosine phosphatase SHP-1 or SHP-2 gene that binds depending on the phosphorylation of the intracellular portion of the BIT protein, and the BIT protein. Neurotrophin (BDNF etc.) and its receptor (Trk B etc.) genes known to be involved in the signal transduction, Src family protein tyrosine kinase (Src, which is involved in phosphorylation of intracellular part of BIT protein) Fyn, etc.) and the like, and other examples include genes of molecules related to signal transduction involving the BIT protein. El2 (Rise), which is also known as an epilepsy-related gene,
M. , Et al. , Science, 253, 669
-673 (1991)) and the like. A non-human animal in which the expression of such plural genes is lost or reduced can also be used for screening of a preventive and / or therapeutic drug for the above-mentioned diseases, etc., and thus the preventive and / or therapeutic treatment of the resulting diseases. Drugs are also included in the present invention.

As a screening method, for example,
The test substance is added to the non-human animal of the present invention or a biological sample obtained from the animal, and B in the animal or biological sample is added.
It can be carried out by comparing a phenotypic change involving the IT protein or a change in the BIT gene expression level with an untreated control animal or a biological sample. Examples of the biological sample include various organs, tissues, cells, extracts thereof, tissue specimens, cultured cells, body fluids such as blood and urine, and the like. The non-human animal of the present invention, for example, can be suitably used as a model of cranial nervous system diseases, immune system diseases, etc., therefore, for analysis of such diseases, a brain slice sample, cranial nerve cells,
Immune system cells and the like are particularly preferably used. Examples of test substances 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.
The amount of the test substance added can be appropriately selected depending on the administration subject, administration method, properties of the test substance, and the like.

Examples of the method for treating the non-human animal of the present invention with the test substance as described above include known methods commonly used such as oral administration and intravenous injection. It may be appropriately selected depending on the properties of the substance. When using a biological sample obtained from the animal, for example, when using cultured cells, it can be performed by a method of culturing by adding a test substance to a culture solution.
Further, for example, when a brain slice sample is used, it can be carried out by a method of directly contacting a test substance prepared in a solution form.

The analysis of phenotypic changes involving the BIT protein can be carried out using, for example, a behavioral analysis method. Specifically, it can be arbitrarily selected from the various behavioral analysis methods described above and used, but for the non-human animal of the present invention, the forced swimming test, Morris water maze test, etc. are preferably used. . It is also preferable to select and use a behavioral analysis method that can detect a significant difference between the animal and the wild-type animal. As a result of such behavioral analysis, for example, the results obtained from the non-human animal of the present invention to which the test substance was administered and the untreated control animal were compared and analyzed, and when a significant difference was obtained, administration was performed. The test substance can be determined to be useful as a therapeutic and / or prophylactic agent for diseases involving the BIT protein.

As another analysis of phenotypic changes, for example,
When the extract or the like of the tissues or cells of the non-human animal of the present invention is used, measurement of the amount of various biological substances, measurement of the activity of biological substances and the like can be mentioned. Further, in the case of cultured cells or tissue specimens, morphological changes, measurement of proliferation rate, measurement of intracellular calcium amount, electrophysiological analysis, measurement of biological substance secretion amount and the like can be mentioned. This analysis can also be determined by comparing the results of the case where the test substance is added and the case where no treatment is performed, as in the above.

When the BIT knockout animal is a heterozygous animal, the change in the expression level of the BIT gene may be analyzed. Such an analysis can be carried out using a known method such as Northern blotting or Western blotting when an extract of tissue or cells is used. In the case of tissue samples such as brain slices, in situ
Analysis can also be performed by the u hybridization method, the immunohistological staining method, or the like. When these analyzes were performed, and when a significant difference was observed between the non-human animal of the present invention and an untreated control animal, or between biological samples obtained from these animals, the administered test substance was It can be determined that the BIT protein is useful as a therapeutic and / or prophylactic agent for diseases associated with it.

Using the method as described above, a significant difference between the BIT knockout animal administered with the test substance and the untreated animal or between the biological sample administered with the test substance and the untreated biological sample was used. When a difference is obtained, the selected substance is further added to a wild-type or hetero-type animal for the purpose of confirming that the obtained significant difference has an effect on the phenotypic change caused by the disappearance of the BIT expression level. Alternatively, it is preferable to add it to a biological sample obtained from these animals and detect the influence of the substance on them. The wild-type or heterozygous animals used for such analysis are preferably litters.

4. Treatment of diseases involving BIT protein
And / or prophylactic drug 3. The substance determined to be useful as a therapeutic and / or prophylactic agent for a disease involving the BIT protein using the screening method of 1. can be used as a safe and low-toxic preventive and / or therapeutic agent for the disease. .
Further, a compound derived from the substance obtained by the above screening method can be used as well. These compounds may form salts, and may be hydrates, solvates and the like. As the salt of the compound, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid,
Examples thereof include pharmaceutically acceptable salts such as salts with organic acids and 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. Preferable examples of the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine,
Examples thereof include salts with ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine and the like. Preferable examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable 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 salt. Preferable examples of the salt with a basic amino acid include salts with arginine, lysine, ortinine, and preferable examples with the acidic amino acid include salts with aspartic acid, glutamic acid and the like.

5. Treatment of diseases involving BIT protein
And / or a method of formulating a prophylactic agent as described in 4. above. Although the substance described in 1) can be administered alone to patients with the above-mentioned diseases, etc., one or more of these active ingredients can be mixed and administered.
Further, it is preferable to formulate the substance into a pharmaceutical composition using a pharmacologically acceptable additive for pharmaceutical preparation and to administer the same. For example, tablets with sugar coating if necessary,
Capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublingual agents, liquids,
It can be used orally as an elixir, emulsion, suspension or the like, or parenterally as an injection prepared as a sterile aqueous or oily solution, a suppository, an ointment, a patch or the like. These include, for example, admixing the substance with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form required for generally accepted formulation practice. It can be produced by a method known in the art such as filling, tableting or tableting. The amount of active ingredient in these pharmaceutical compositions is such that a suitable dose within the indicated range can be obtained.

For example, as additives that can be mixed with tablets, capsules and the like, binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, cellulose, mannitol, lactose, etc. Fillers, cornstarch, gelatin, alginic acid, etc., swelling agents, starch, polyvinylpolypyrrolidone, starch derivatives, disintegrating agents such as sodium starch glycolate, lubricants such as magnesium stearate, wetting agents such as sodium lauryl sulfate, A sweetening agent such as sugar, lactose or saccharin, a flavoring agent such as peppermint, red oil or cherry, and the like are used. Further, the tablet may be coated with an enteric coating agent or the like, if necessary. In the case of capsules, liquid carriers such as fats and oils may be contained in addition to the above additives.

Further, for example, a sterile pharmaceutical composition used as an injectable solution is prepared by treating the active substance in a vehicle such as water for injection,
It can be formulated in accordance with usual practice such as dissolving or suspending naturally occurring vegetable oils such as sesame oil and coconut oil. As the aqueous solution for injection, for example, physiological saline,
Isotonic solutions (D-sorbitol, D-mannitol, sodium chloride, etc.) containing glucose and other adjuvants are used, and suitable solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene, etc.) are used. Glycol, etc.), nonionic surfactants (Polysorbate 80TM, HCO-50, etc.) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate which is a solubilizing agent,
It can be used in combination with benzyl alcohol and the like. Also,
Examples of the above pharmaceutical composition include buffers (phosphate buffer, sodium acetate buffer, etc.), soothing agents (benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (human serum albumin, polyethylene glycol, etc.). ), Preservatives (benzyl alcohol, phenol, etc.), and antioxidants may be added. The thus-prepared aqueous liquid for injection is usually produced by dissolving the substance in a medium as described above, sterilizing by filtration, and filling in a suitable sterilized vial or ampoule. The water in the vial may be removed under vacuum after freezing the pharmaceutical composition to increase stability. An oily liquid can be produced in substantially the same manner as an aqueous liquid, but it can be suitably produced by suspending the compound in the above medium and sterilizing with ethylene oxide or the like.

Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be administered, for example, to humans or non-human animals as a prophylactic or therapeutic drug for the above-mentioned diseases and the like. The dose of the compound contained in the composition can be appropriately determined and used depending on the target disease, symptom, target organ, administration subject, administration method and the like. Further, it is desirable to administer the daily dose in one to several divided doses.

6. Other Uses of Non-Human Animal of the Present Invention Since the non-human animal of the present invention has a BIT gene expression level that is lost or reduced as compared to the wild type, various biological activities produced in vivo by the BIT protein are deleted. Yes, B
It can be used for analysis of various functions of IT proteins. For example, since the BIT protein has an important function in the cranial nervous system, a brain slice sample or a cultured cranial nervous system cell obtained from the animal can be used for analysis of cranial nervous system function. Moreover, since the BIT protein also plays an important role in the immune system, immune system cells such as macrophages and T cells obtained from the animal are used for analysis of the immune system function. It is also possible to analyze interactions, phagocytosis, macrophage fusion mechanism, etc. using macrophages and dendritic cells. The BIT gene is
Since it has a polymorphism in the nucleotide sequence site encoding the V domain involved in antigen recognition, it is useful for analysis of diversity such as individual difference and species difference in the analysis of the above-mentioned cranial nervous system function and immune system function. is there.

Furthermore, the BIT protein has an amino acid sequence very similar to the major histocompatibility antigen known to cause the strongest rejection reaction in transplantation of organs and the like, and the V domain involved in antigen recognition has major tissues. It has been elucidated that they also have polymorphisms similar to compatible antigens (Sano S., et al., Biochem.
J. , 334, 667-675 (1999)). It is known that the rejection mechanism that occurs during transplantation is extremely complicated by the involvement of various minor histocompatibility antigens in addition to the strongest rejection by major histocompatibility antigens. Since the non-human animal of the present invention is also considered to play a part in that, it can be used for elucidation of the mechanism of the rejection reaction occurring during transplantation. In addition, the non-human animal of the present invention in which the major histocompatibility antigens match and the expression level of the BIT gene disappears or decreases, preferably disappears is used as a donor (donor), and organs obtained from the animal or By using the tissue for transplantation, the rejection reaction in the recipient (patient) can be suppressed and the engraftment of the transplanted organ or tissue can be improved.

[0048]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. Example 1. Construction of Targeting Vector (1) Preparation of Partial DNA Fragment of Mouse BIT Gene Clone # 4 (Sano S., et a, containing the first exon of BIT gene cloned from mouse 129 / SvJ genomic library (Stratagene)).
l. , Biochem. J. , 334, 667-675
(1999)), EcoRI treatment was carried out to cut out a fragment of about 7.5 kb containing the first exon, and then pBlue
pBS # 4-1 by subcloning into scriptII (pBSII) KS (-) (manufactured by Stratagene).
It was set to E.

A PCR method using pBS # 4-1E as a template and sense primer GA-F (SEQ ID NO: 1), antisense primer GA2-R (SEQ ID NO: 2) and LA Taq (Takara Shuzo). As a result, a DNA fragment of about 4.1 kb on the 5'side from the translation initiation point of the BIT protein was obtained. This DNA fragment contains an N at the 5'end from the primer sequence.
An otI site and an EcoRI site were introduced at the 3'end.
This DNA fragment was designated as pCRII (Invitrogen)
Subcloned into pCRGA.

Similarly, PCR was performed using pBS # 4-1E as a template and the sense primer GB-F (SEQ ID NO: 3) and the antisense primer GB-R (SEQ ID NO: 4).
By the method, DN of about 3.1 kb on the 3'side of the first exon
A fragment was obtained. This DNA fragment had a SalI site at the 5'end as a genomic sequence depending on the sequence of the primer.
XhoI site, HindIII site, Kpn at 3'end
It was confirmed that the I site was introduced. This DNA fragment was subcloned into pCRII and named pCRGB.

(2) DN containing neomycin resistance gene
Preparation of A fragment As a DNA fragment containing a neomycin resistance (neo) gene, a neo cassette "pKJ2" (Boer) in which a neo gene and a PGKpolyA signal are linked downstream of a phosphoglycerate kinase (PGK) promoter is used.
P. H. , Et al. , Biochemical
Genetics, 28, 299-308 (199
0)) was used.

(3) DN containing thymidine kinase gene
Preparation of A fragment A DNA fragment containing the thymidine kinase (TK) gene is
MC1 promoter (Thomas KR and
Capecchi M. R. , Cell, 51, 503
-512 (1987) downstream of the herpes simplex virus thymidine kinase gene (Manso
ur S. L. , Et al. , Nature, 33
6,348-352 (1988) ligated,
The MC1-TK cassette "F3" cloned into the EcoRI / EcoRV sites of pBSIIKS (-) was used. MC1-TK was excised by treating F3 with BamHI and SalI, and the fragment was blunted with S
MC1-T by adding alI linker (manufactured by Toyobo Co., Ltd.)
A SalI site was introduced at the 5'end of K. Then Sal
Treated with restriction enzymes I and HindIII, pBSIIKS
It was subcloned into (-) and named pBSTK.

(4) Preparation of targeting vector NotI / Kp of pBSIIKS (-) was prepared as follows.
At the nI site, D on the 5 ′ side from the translation initiation point of the BIT protein
NA fragment, PGK-neo fragment, 3'from the first exon
The DNA fragment on the side and the MC1-TK fragment were ligated in order and cloned to prepare a targeting vector.
First, a NotI-EcoRI fragment obtained by treating pCRGA with NotI and EcoRI was cloned into the NotI / EcoRI site of pBSIISK (−), and this was named pBSGA. Next, EcoRI of pBSGA
The PGK-neo fragment obtained by treating pKJ2 with EcoRI and XhoI was cloned into the / XhoI site, and named pBSAn. Furthermore, pCRGB was combined with SalI and K at the XhoI / KpnI site of pBSAn.
The SalI-KpnI fragment obtained by treating with pnI was cloned and named pBSAnB. Finally, pBSA
Salmon pBSTK at the XhoI / KpnI site of nB
MC1 obtained by treatment with I and HindIII
-TK fragment was cloned and it was decided to use this as a targeting vector. FIG. 1 shows a construction diagram of the targeting vector constructed as described above. In the figure, ATG represents the translation initiation point of the BIT protein. E is EcoRI, B is BamHI, H is HindI
The restriction enzyme site of II is shown.

Example 2. Targeting to ES cells
Introduction of vector (1) The homologous recombination targeting vector in ES cells was linearized by restriction enzyme treatment with NotI and purified to prepare 1 × 10 ⁸ ES cells (E1
4TG2a; Hooper M .; , Et al. , Na
true, 326, 292-295 (1987)),
Electroporation method (1000 V, 3 μ, at a concentration of 94 μg / 1.7 × 10 ⁷ cells)
F). GenePulser (manufactured by Bio Rad) was used for electroporation. 175 μg / ml G418 and 2 μM FIAU
Was added to the medium to obtain colonies. The medium is GM
EM (manufactured by GIBCO), 10 ⁻⁴ M β-mercaptoethanol (manufactured by Sigma), 24 mM NaHCO 3
₃ (manufactured by Sigma), 1 mM sodium pyruvate (GI
BCO), 0.1 mM non-essentia
l amino acids (manufactured by GIBCO), 10
% FCS, 1000 units / ml LIF (ESG
RO, manufactured by AMRAD) was used. ES
The cell E14TG2a strain is derived from a 129 strain mouse,
Austin G. from Edinburgh University. Granted by Dr. Smith.

(2) Selection of homologous recombinants 300 were picked up from the obtained colonies,
It was cultured and attached to a 96-well plate. The cells attached to the 96-well plate were washed twice with PBS, placed at −20 ° C. overnight, 100 μl of H ₂ O was added, and the cells were incubated at 70 ° C. for 10 hours.
Processed for minutes. Next, Proteinase K (1 mg /
(2 ml) was added, the mixture was incubated at 37 ° C for 1 hour, and then treated at 95 ° C for 5 minutes. By using this as a template DNA and using sense primer Neo-F (SEQ ID NO: 5), antisense primer G4-R (SEQ ID NO: 6), and LA Taq for detecting the introduced neo gene, PCR was performed. Screened. As a result, 5 homologous recombinants in which the BIT gene was knocked out were obtained.

Furthermore, DNA from these homologous recombinants
Was prepared and analyzed by Southern blotting using four types of probes, and it was confirmed that it was definitely a homologous recombinant. As the probe I, a DNA fragment of about 6 kb obtained by subjecting the clone # 4 obtained from the genomic library to a restriction enzyme treatment with NotI and EcoRI was subcloned into pBSIIKS (−),
This was designated as BS # 4-2NE, and the PvuII-EcoRI fragment of about 1.6 kb obtained from this pBS # 4-2NE was used. As the probe II, BALB / c mouse BIT cDNA (GenBank Accessio) was used.
n No. The DNA fragment having the sequence of base numbers 1-303 obtained from D85785) by the restriction enzyme treatment of EcoRI-NcoI was used. As the probe III, an ApaI-NcoI fragment of about 1.5 kb obtained from pBS # 4-1E was used. As the probe neo, Pst of about 600 bp obtained from the above pKJ2 was used.
The I-BamHI fragment was used. Southern blot analysis
According to "User Guide for Hybridization Using DIG System" (Boehringer Mannheim), the DNA treated with the restriction enzyme is subjected to agarose gel electrophoresis, transferred to a nylon membrane, and then DIG.
High S containing probe labeled with DIG using DNA labeling kit (Boehringer Mannheim)
After hybridization with a DS concentration hybridization buffer, an alkaline phosphatase-labeled anti-digoxigenin antibody (Boehringer Mannheim), CSPD (disodium 3- (4-mesoxyspiro) {1,2-dioxycetane-3,2 '-(5 '-Chloro) tricyclo [ ^3.3.1.1,7 ] decane} -4-
Il) Phenylphosphoric acid; 11.6 mg / ml (25 m
M) solution: Boehringer Mannheim) (Sano S., et al., Biochem.
J. , 334, 667-675 (1999)).

Example 3. BIT knockout mouse work
Ltd. (1) Preparation chimeric mice of chimeric mice, phase clones homologous recombination to cause ES cells were picked up again from a colony obtained in above (1), blastocysts C57BL / 6N mice were pseudopregnant induced It was generated by transplantation using the method of injecting into. Chimeric mouse male and wild type C57BL / 6N produced
The genotype of the resulting offspring was analyzed by Southern blotting and PCR to cross the germ line (germ-line transm).
confirmation).

(2) Mating and Rearing of Mice In order to purify the genetic background, male heterozygous mice were crossed with female C57BL / 6N strains for backcrossing. 6 times backcrossing once, 12 times backcrossing twice, female heterozygous mice with male C5
The Y chromosome was derived from the C57BL / 6N strain by mating 7BL / 6N strain mice. The animals were raised at 8:00 for 2 hours with lights on at 20:00 and off for 2 hours at room temperature of 23.0 ± 1 ° C. at a specific pathological level by regular monitoring of microorganisms.
e (SPF).

For the routine identification of genotypes (homotype, heterotype, and wild type), two types of analysis by the PCR method using the DNA extracted from the tail as a template were carried out as follows,
It was confirmed that the same result was obtained by both methods. First, as a first method, a sense primer (neo-F; SEQ ID NO: having a partial base sequence of neo gene) is used.
5) and a primer (RT-F1; SEQ ID NO: 8) having a partial base sequence of a normal BIT gene, and an antisense primer (KO- having a partial base sequence of an intron 3 ′ downstream of the first exon of BIT gene). R; PCR was performed in combination with R: SEQ ID NO: 7) to confirm the presence of the BIT gene in which homologous recombination occurred and the normal BIT gene. That is, about 1 mm of the tip of the obtained offspring was cut into a 1.5 ml tube (-20 ° C).
It can also be stored), 100 μl of H ₂ O was added, and the mixture was treated at 80 ° C. for 10 minutes and cooled on ice, and then 1 mg / m 2.
Add 2 μl of Proteinase K to 37 ° C
And incubated for 1 hour. Add this to 99 ℃
After treatment for 5 minutes at 4 ° C., PCR was carried out using 3 kinds of primers at the same time as the template DNA stored at 4 ° C. The homo type gave a product of about 450p with the primer Neo-F (SEQ ID NO: 5) and the primer KO-R (SEQ ID NO: 7), and the wild type had a primer RT-F1 (SEQ ID NO: 8).
And the primer KO-R (SEQ ID NO: 7) to detect a 262 bp product. Further, a heterotype having both genotypes was identified by detecting the above-mentioned two PCR products (FIG. 2).

As another method for identifying the genotype by the PCR method, the fact that the BIT gene of the BIT knockout mouse is derived from the 129 line (ES cell) is used.
The analysis was performed by detecting the difference (polymorphism) from the C57BL / 6N system in the nucleotide sequence encoding the V type domain of IT protein. First, using the DNA from the tail obtained as described above as a template, the primer GV-F (SEQ ID NO:
PC with 9) and primer GV-R0 (SEQ ID NO: 10)
R was performed and the product was cleaved with the restriction enzyme KpnI. In the wild type, the partial base sequence encoding the V domain of the C57BL / 6 system-derived BIT protein amplified by PCR using such a primer remains 491 bp and is not cleaved, but in the homotype, it is derived from the 129 system. Partial base sequence is 3
It could be confirmed because it was cut into 23 bp and 168 bp. The heterotype was confirmed by detecting three fragments, an uncut fragment and a cut fragment (Fig. 3).

(3) BIT by Western blotting
Expression analysis of proteins Genotyped BIT knockout mouse heterozygous
Type, homozygous and wild type mice (6 backcrosses, 3
Brain and spleen, and homogenate each
Iz buffer (2% SDS, 2% β-mercaptoetha
Nol, 20 mM Tris-HCl (pH 7.5),
0.1 mM PMSF, 500 U / ml aprotinin,
10 μg / ml leupeptin, 1 mM EDTA, 1 m
M Na ₃VO_Four) In Teflon® homogeny
Homogenized by the. Protein of the obtained extract
After quantifying the concentration, 50 μg protein was added to SDS-PA.
Suspended in GE sample buffer and boiled for 3 minutes. Sun
Pulls were separated by 4-20% SDS-PAGE
After that, transfer to PVGF membrane and add 5% skim milk / PBS.
Blocked. Anti-BIT protein serum (Sano
S. , Et al. , Biochem. J. , 334,
667-675 (1999)) overnight, and then
Goat anti-rabbit IgG conjugated with oxidase (Zymed)
ECL (Amersham Pharma)
(Manufactured by SIA) (Sano S., eta)
l. , Biochem. J. , 334, 667-675
(1999)). Figure 4 shows wild type (+ / +) and hetero (+)
/-), Homozygous (-/-) mouse brain (Brain)
And Western blot using spleen (Spleen)
The results are shown. The expression of BIT protein in the brain and spleen is
Not detected in homozygous mice and wild in heterozygous mice
It was found that an intermediate amount of type mice and homozygous mice was detected.
It was

(4) Src by Western blotting
Expression analysis of family The expression analysis in the BIT knockout mouse was performed for the Src family, which is a protein tyrosine kinase that is considered to phosphorylate intracellular tyrosine residues of BIT protein. Src, Fyn, Lck, and Lyn, which are molecules known as the Src family, were analyzed in the same manner as the Western blot analysis of the BIT protein performed in (3) above. Antibodies include anti-Sr
c antibody Ab-1 (manufactured by CALBIOCHEM), anti-Fy
n antibody (TransductionLaborator
ies), anti-Lck antibody 3A5 (Santa Cr
uz Biotechnology), anti-Lyn antibody H-6 (Santa Cruz Biotechnology).
(manufactured by LOGY) was used. As a secondary antibody, peroxidase-conjugated goat anti-mouse IgG antibody (Jacson
Laboratories) was used. as a result,
It was found that the amounts of Fyn and Lck were significantly reduced in the spleens of homozygous mice (Fig. 5). This result indicates that T cells are abnormal, and it was found that BIT knockout mice have abnormal immune system function.

(5) Measurement of body weight of BIT knockout mouse The body weight of BIT knockout mouse was measured at 4 weeks, 32 weeks and 44 weeks of age. It was measured and analyzed using homozygous, heterozygous, and wild-type mice of each age. The analysis results are shown in Table 1. As a result, the weight of homozygous mice tended to be lighter than that of heterozygous mice and wild-type mice. As a result of analysis by the least significant difference method after one-way analysis of variance, at 4 weeks of age, there was no significant difference in body weight between female and male, but at 32 weeks of age, between female homozygous mice and heterozygous mice ( p = 0.041), and in males between homozygous and wild type mice (p = 0.000).
2), and between homozygous mice and heterozygous mice (p = 0.
A significant difference was seen in 0037). Moreover, at 44 weeks of age, between female homozygous and wild type mice (p <
0.0001), between homozygous mice and heterozygous mice (p <0.0001), between homozygous mice and wild type mice (p <0.0001), between homozygous mice and heterozygous mice. (P <0.0001) between wild-type mice and heterozygous mice (p = 0.0351). From these results, it was found that the weight difference between the BIT knockout mouse and the wild-type mouse became clear after a while after becoming an adult animal. Especially in female homozygous mice, it was found that the weight gain after 32 weeks was significantly slowed down.

[0064]

[Table 1]

Example 4. BIT knockout mouse line
Dynamic analysis For behavioral analysis, C57BL / 6N mice were backcrossed 6 times to obtain male mice [14 wild-type mice,
16 homozygotes] were used. In the forced swimming test using the automatic measurement device, male males [8 wild type, 8 hetero type, 9 homo type] obtained as a result of backcrossing 12 times were used. The analysis was performed between 4 and 15 months of age. Before analysis, the animals were bred in one cage / one cage, and handled for five days (three times a minute for one day) to fully familiarize the experimenter with the experiment. The experiment was performed between 13:00 and 18:00.

The results obtained by each behavior analysis were statistically analyzed. Open field test,
For the light-dark selection test and the electric shock sensitivity test, t-test was performed. For others, after the one-way analysis of variance or two-way analysis of variance, the least significant difference method (Fisher's least) was calculated between the paired groups.
SIGNIFICANT DIFFERENCE (LS
D) test) was performed. Values are shown as mean ± standard error. In these analyses, a p value of less than 0.05 was determined to be "significantly different." Statistical analysis
Application software StatView J (Ab
acus Concepts).

(1) Open field test The open field test, which is an index of the spontaneous activity of the mouse, was performed by Ikegami S. et al. Behav. Bra
in Res. , 65, 103-111 (1994). The amount of locomotor activity and the standing response of the mouse were measured by the open field box (32 × 3
2 × 20 cm) was used to measure in a soundproof room. The open field box has an infrared sensor (7x7
2 cm and 4.5 cm from the floor), the locomotion activity amount was detected by the lower sensor, the rising reaction was detected by the upper sensor for 30 minutes, and recorded by the computer. The measurement results of the average locomotor activity and standing up response for 30 minutes are
1688.1 ± 87.7 in wild type mice,
218.4 ± 20.3, 1481.8 in homozygous mice
The values were ± 63.9 and 171.6 ± 13.5. Although a t-test was performed, no significant difference was found in the locomotor activity (p = 0.063) and the rising reaction (p = 0.059).

(2) Rod crossing balance sensation test A rod crossing balance sensation test, which is an index of motor coordination, was performed. The mouse was placed in the center of a square bar (15 mm on a side, 60 cm in length), and it was tested whether or not the mouse could be crossed without falling to the end (bar installation height: 46 cm). As a result, no abnormality was found in homozygous mice in this test.

(3) Wire hanging muscle strength test A wire hanging muscle strength test, which is an index of muscle strength, was performed.
A mouse was hung in the center of a wire (diameter 0.9 mm, length 60 mm) to test whether it could hang for 10 seconds or more (wire installation height 43 cm). As a result, no abnormality was found in homozygous mice in this test.

(4) Light / dark selection test The light / dark selection test, which is an index of fear reaction and anxiety, is Ike.
gami S. , Et al. , Hum. Mol. Gen
et. , 8, 1409-1415 (1999). Place the open field box in a bright room (16 x 32 x 20 cm) and half in a dark room in a soundproof room with fluorescent lights turned off.
Illuminated with a light bulb. 7 holes (3 x
9 cm), which allows the mouse to pass through, and the infrared sensor beam passes through. The mouse was first placed in the center of the light room and recorded with a video camera for 15 minutes.
When four limbs of the mouse moved to the next room, it was considered that the room had moved, and the percentage of time in the light room was calculated. as a result,
The average percentage of time spent in a bright room for 15 minutes was 42.3 ± 2.1% in wild type mice and 42. ± 2.1% in homozygous mice.
It was 1 ± 2.6%. No significant difference was found in the t-test (p = 0.96).

(5) Fear Conditioning Test Context-dependent fear conditioning test in which place stimulation causes a freezing reaction in association with electric shock.
A test of "fear conditioning" was performed. Ikegami S. , Et al. , Hum. M
ol. Genet. , 8, 1409-1415 (199
It was performed by the method described in 9). Conditioning device (20 x 20
The floor (× 20 cm) is made of a stainless steel rod, and an electric shock can be applied with a shock scrambler (SGS002; manufactured by Muromachi Kikai Co., Ltd.). The device was placed in a soundproof room and observed and recorded from the adjacent room with a video camera. The shock scrambler and controller were operated by a remote switch from the next room.

The mouse was placed in the apparatus, and after 2 minutes and 30 seconds, 0.5 mA was applied from the floor for 1 second 3 times at 1 minute intervals. After completion of the electric shock, it was left for 1 minute and then returned to the cage. Twenty-four hours after the electric shock, the mouse was placed in the apparatus again, and the freezing (Fr
easing) reaction was measured. Freezing was based on the fact that there was no movement other than breathing. Whether freezing or not was determined every 5 seconds, and the percentage (%) of freezing time per observation time was obtained every 1 minute. The freezing reaction was measured in the same manner 7 days after the electric shock was given. When the ratio (%) of the average freezing time per minute was analyzed by two-way analysis of variance,
Between wild-type and homozygous mice, [F
(1,140) = 3.05, p = 0.83], and after 7 days, [F (1,40) = 0.01, p = 0.92],
Neither was a significant difference.

(6) Electric Shock Sensitivity Test The electric shock sensitivity test for analyzing the sensitivity to electric stimulation is based on Ikegami S. et al. , Et al. , Hu
m. Mol. Genet. , 8, 1409-1415
It was performed by the method described in (1999). The mouse was placed in the conditioning device and 0.05, 0.08, 0.1, 0.
In the order of 2, 0.3, 0.4, 0.5, 0.6, and 0.8 mA, an electric shock of 1 second is given at intervals of 10 seconds, screaming (vocalization), and jumping (jump).
ing), the minimum stimulus intensity that causes running is examined. After the test, scream (voca
lization), jumping,
For each running, the minimum stimulus intensity (mA) was analyzed by t-test, and screaming [p = 0.88], jumping up
[p = 0.85] and running around [p = 0.67], showing no significant difference between wild-type and homozygous mice.

(7) Morris's Water Maze Test The Morris's water maze test, which is an index for spatial learning, is described by Ikegami S. et al. , Et al. , Hu
m. Mol. Genet. , 8, 1409-1415
It was performed by the method described in (1999). In a circular pool (diameter 120 cm), a 10 cm diameter platform on which a mouse rides is immersed 0.5 cm in water (25 cm).
(° C.), skim milk was added to make the water cloudy.
A CCD camera was attached above the center of the pool and connected to a color video tracking system (CAT10; Muromachi Kikai Co., Ltd.) to trace and record the locus of the mouse.
A computer recorded the time taken for the mouse to reach the platform, the migration distance, and the migration trajectory.

The experiment was conducted for 7 days. From the 1st to the 4th day, using a hidden platform (located in the center of one of the four quarters of the pool)
Six trials a day were made, and the task of riding on the platform was performed at intervals of 30 minutes or more (platform learning). 30 if you can get on the platform within 2 minutes
After spending seconds on the platform, they were returned to their cages. Two
Mice that could not ride within a minute were put on the platform at the time of 2 minutes and left for 30 seconds. The starting positions for swimming were arbitrarily selected from the eight places set at equal intervals around the pool, excluding the position closest to the platform, so that it could be once from the same place in one day. After the last trial on the 4th day, the platform was removed, the mice were allowed to swim for 1 minute and the time to swim in each of the 4 compartments was recorded automatically (transfer test).
On the 5th day and the 6th day, the platform was placed in the opposite compartment from the 1st day to the 4th day, and 6 trials a day were performed for 2 days (reverse learning). After the final trial on day 6, the platform was removed, the mouse was allowed to swim for 1 minute and the time to swim in each of the 4 compartments was recorded automatically (inversion transfer test). 7
On the day, 10 × so that you can see the position of the platform
A white flag of 10 cm was erected and the platform position was changed each time, and 6 trials were performed (visible test).

When such a test was carried out, many homozygous mice were found to float during the test without swimming. Therefore, we first analyzed the data considered to be established as a learning task, except for the floating mouse data. In platform learning, wild type mice (n = 10) and homozygous mice (n = 10)
= 6) in the time to reach the platform of 2)
Significant difference in one-way analysis of variance [F (1,276) =
4.01, p = 0.046] was found, and it was found that mild learning dysfunction was observed in homozygous mice. In the reverse learning and the visible test, [F (1, 18
4) = 0.025, p = 0.87] and [F (1,92)
= 0.014, p = 0.91], and no significant difference was found between the wild type mouse and the homozygous mouse. In the transfer test and the reversal transfer test, [F (1,56) = 0.014, p = 0.91] and [F (1,56) = 0.19, p = 0.89], respectively, and one element No significant difference was found by the ANOVA.
Taken together, these results indicate that homozygous mice have a very mild impairment of learning function.

Next, the data of the transfer test and the reversal transfer test were analyzed for the swimming time and swimming distance, including the data of the mouse that floated without swimming. The swimming time was [F (3,55) = 6.1 by one-dimensional ANOVA between wild-type and homozygous mice.
5, p = 0.011], and even when analyzed by the least significant difference method (LSD), it was revealed that the migration time of the homozygous mice was obviously shorter than that of the wild-type mice ( Transfer test; p = 0.0037, reverse transfer test; p = 0.0037) (Fig. 6A).
Similarly, for the swimming distance, one-dimensional ANOVA between wild type mice and homozygous mice [F (3,55) = 8.
92, p <0.001], and further analyzed by the least significant difference method, it was found that the homozygous mice had a significantly shorter migration distance than the wild-type mice (transfer test; p = 0.0009, reversal transfer test; p = 0.0004) (FIG. 6B).

(8) Forced Swimming Test In the Morris water maze test performed in (7) above, knockout mice tended to float without swimming, so next, wild type mice, heterozygous mice,
It was decided to perform a forced swimming test using homozygous mice for further detailed analysis. The Forced Swim Test is the test when an animal is forced to swim in an unavoidable tank.
It measures the time to show an immobility state (immobility time).

First, a transparent water tank (diameter 20 cm, height 2
Water (25 ° C.) was put in 5 cm) to a depth of 14 cm, and an infrared sensor system (SuperMex Sensor PYS-001; manufactured by Muromachi Kikai Co., Ltd.) was attached 20 cm above the water surface. The infrared sensor incorporating this multi-Fresnel lens can measure the spontaneous motor activity by detecting the infrared rays emitted from the animal.
Infrared sensor power unit for sensor (MPU-
8A; Muromachi Kikai Co., Ltd.) and a data recording interface (DI-064W; Muromachi Kikai Co., Ltd.) were connected and recorded by controlling with a computer. The immobility time was measured by placing the mouse in an aquarium and determining whether or not the immobility time was determined for each second depending on whether or not the spontaneous movement was measured every one second.

On the first day, in the two-dimensional analysis of variance, the difference in immobility time among the wild-type mouse, the heterozygous mouse, and the homozygous mouse was [F (2,330) = 23.04, p <0. ．
0001], and when analyzed by the least significant difference method, homozygous mice were wild type mice (p <0.0001).
It was found that the immobility time was significantly longer than that of the heterozygous mice (p <0.0001) (FIG. 7A). Even in the test on the second day, in the two-dimensional analysis of variance, the difference in immobility time between mice of each genotype was [F (2,330) = 18.1.
7, p <0.0001], and when analyzed by the least significant difference method, homozygous mice were wild-type mice (p <0.0001) and heterozygous mice (p = 0.001).
It was shown that the immobility time was significantly longer than that of 0041) (FIG. 7B). Furthermore, it was also shown that the heterozygous mice had a significantly longer immobility time than the wild-type mice (p = 0.0025). From these results, it was found that when the expression level of the BIT protein disappeared or decreased, the immobility time in the forced swimming test increased. BIT
Immobility time in knockout mice was most markedly short during the first 5 minutes on day 2.

The forced swimming test is a behavioral analysis method used for screening antidepressant drugs and attracting attention for its relationship with depression. BIT knockout mice (homo type and hetero type) showed a long immobility time in the forced swimming test, and it was found that emotions associated with depression were abnormal.
It was shown to be useful as a model for depression and the like.

[0082]

INDUSTRIAL APPLICABILITY The non-human animal of the present invention in which the expression level of BIT gene disappears or is lower than that of the wild type is useful as a model animal for diseases such as depression associated with BIT protein. The animal can be used to screen for a therapeutic and / or prophylactic drug for the disease.

[0083]

[Sequence list] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation <120> BIT knock out non-human animal <130> J07924 <140> <141> <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 1 tagttctaga gcggccgctc aaatcttgac tacttgggga ccgaacagct a 51 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 2 caggaattcg tcgaccgggc ggcgcgggcg aacgactaa 39 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 3 ctaatgtcga caaatccctg taacgggggt tacagatgca 40 <210> 4 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 4 cgcggtacca agcttctcga gcttactcct ggaacaagtt ttggactcag tttt 54 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 5 tcctcgtgct ttacggtatc gccgctcccg att 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 6 tgggctgcat acgcctaagt cactcactcc tct 33 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 7 caagtggctg accctctctg ggcctcaact 30 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 8 tcaccgccgc tctccctcct tgctctgca 29 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 9 tgagcctgca ggatccctta aggttagtca 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 10 agccacactt acgagatcca agatgttatt 30

[Brief description of drawings]

FIG. 1 is a construction diagram of a targeting vector used for producing the non-human animal of the present invention. In the figure,
ATG represents the translation start point of the BIT protein. E is Eco
RI and B indicate BamHI and H indicates HindIII restriction enzyme sites.

FIG. 2 shows the genotype of BIT knockout mice
It is a photograph of electrophoresis showing the results of analysis by the CR method. In the figure, + / + is a wild type, +/- is a hetero type, and-/-.
Indicates a homozygous type.

FIG. 3 shows the genotype of BIT knockout mice as B
2 is an electrophoretic photograph showing the results of analysis by PCR using a primer capable of detecting a V domain polymorphism of IT protein. + / +, +/-, and-/-in the figure have the same meaning as in FIG.

FIG. 4: Anti-BIT using brain and spleen extracts of wild-type, heterozygous and homozygous BIT knockout mice.
It is the result of having analyzed the expression of BIT protein by Western blotting using protein serum. + / +, + / In the figure
-,-/-Are synonymous with FIG.

FIG. 5: Using the extracts of brain and spleen of wild-type, heterozygous and homozygous BIT knockout mice, Src,
It is the result of having analyzed the expression of each protein of Fyn, Lck, and Lyn by Western blotting. + / +, + In the figure
/-And-/-are synonymous with FIG.

FIG. 6: BI using Morris water maze test
It is a graph of the result of having performed the behavioral analysis of T knockout mouse and a wild-type mouse. FIG. 6A shows the result of the experiment of migration time, and FIG. 6B shows the result of the experiment of migration distance. + / + And-/-in the figure are shown in FIG.
Is synonymous with.

FIG. 7 is a graph showing the results of behavioral analysis of BIT knockout mice and wild-type mice using a forced swimming test. FIG. 7A shows the experimental results on the first day, and FIG. 7B shows the experimental results on the second day. + / +, +/-, and-/-in the figure have the same meaning as in FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/15 C12N 15/00 A 33/50 5/00 BF term (reference) 2G045 AA29 AA40 BA13 BB20 CB01 CB17 DA12 DA13 DA14 DA44 FB03 FB05 4B024 AA01 AA11 BA80 CA05 CA06 CA09 CA20 DA02 EA04 GA14 GA18 GA27 HA13 HA14 4B065 AA91X AB01 AC20 BA03 BA16 BA24 BB01 BC01 CA46 4C084 AA17 NA14 ZA12

Claims (11)

[Claims] 1. A non-human animal in which the expression level of BIT gene is reduced or reduced compared to the wild type, or a progeny thereof. 2. The animal according to claim 1, wherein the loss of BIT gene expression is due to disruption of the BIT gene at two BIT loci on the homologous chromosome. 3. The animal according to claim 1, wherein the decrease in the amount of BIT gene expression is due to the disruption of the BIT gene at one BIT locus on the homologous chromosome. 4. The disruption of the BIT gene is caused by the gene DNA.
2 or 3 caused by deletion of the start codon of
The animal described in. 5. The animal according to any one of claims 1 to 4, wherein the animal has a depression-like phenotype. 6. The method according to claim 1, wherein the animal is a rodent.
5. The animal according to any one of 5. 7. The rodent is a mouse.
The animal described in. 8. A test substance is added to the animal according to any one of claims 1 to 7 or a biological sample obtained from the animal,
A method for screening a therapeutic and / or prophylactic drug for a disease involving a BIT protein, which comprises detecting the effect of a test substance on the animal or biological sample. 9. The method according to claim 8, wherein the disease associated with the BIT protein is depression. 10. A BIT containing a substance selected by the screening method according to claim 8 as an active ingredient.
A therapeutic and / or prophylactic agent for diseases involving proteins. 11. A method for formulating a therapeutic and / or prophylactic agent for a disease involving BIT protein, which comprises selecting a substance by the screening method according to claim 8 and formulating the substance.