JP2007000028A - New non-human animals - Google Patents

Abstract

An object of the present invention is to provide a non-human animal in which the expression level of vesl-1S / 1M gene has disappeared or decreased compared to the wild type and a method for producing the same.
In order to destroy the vesl-1S / 1M gene by deleting the exon region specific to the vesl-1S gene and inhibiting splicing to the exon specific to the vesl-1M gene, Construction of a targeting vector for introducing cDNA containing exons 7 to 11 unique to the vesl-1L gene into an alternative 5 'splicing donor site present in the exon is introduced into ES cells for homologous recombination. Then, by crossing the chimeric mice produced by the ES cells, mice with the expression level of vesl-1S / 1M gene disappeared or decreased were provided.
[Selection figure] None

Description

  The present invention relates to a non-human animal or its progeny in which the expression level of vesl-1S / 1M gene has disappeared or decreased compared to the wild type, and a production method thereof.

vesl-1S (also called homer-1a) is a gene isolated as a gene whose expression increases with convulsions and long-term potentiation (LTP) of the hippocampus (for example, see Non-Patent Document 1). reference). vesl forms a family consisting of three genes, vesl-1, vesl-2, and vesl-3. Of these, the vesl-1 gene transcribes three splicing isoforms vesl-1S, vesl-1M (also called ania-3), vesl-1L (also called homer-1c) mRNA in the rat brain To do. The vesl-1 RNA has 11 exons, the vesl-1S mRNA is the 1st to 5th exons, the vesl-1M mRNA is the 1st to 6th exons, and the vesl-1L mRNA is the 1st to 5th and 1st exons. It consists of 7-11 exons. The sequence and structure of the genomic DNA of vesl-1 is described in Non-Patent Document 2 (GenBank registration code NW — 000085, GI: 20909471). The fifth exon of vesl-1S is not spliced at the alternative 5 'splicing donor site described below, and has 11 amino acids added to the C-terminus. That is, the region corresponding to the C-terminal 11 amino acids of the fifth exon is specific for the vesl-1S gene, the sixth exon is specific for the vesl-1M gene, and the seventh to eleventh exons are specific for the vesl-1L gene. Only vesl-1S and vesl-1M induce neural activity-dependent expression, and vesl-1L, vesl-2, and vesl-3 are constitutively expressed (see, for example, Non-Patent Document 3). Hereinafter, in the present specification, the genes of the three splicing isoforms of vesl-1 (vesl-1S, vesl-1M, vesl-1L) are also referred to as “vesl-1S / 1M / 1L gene” and the three splicing isoforms. The forms of RNA are collectively referred to as “vesl-1S / 1M / 1L”, and the proteins encoded by the genes or RNAs of these three splicing isoforms are also referred to as “Vesl-1S / 1M / 1L protein” or “Vesl -1S / 1M / 1L ". In addition, the two splicing isoforms (vesl-1S, vesl-1M) of the three splicing isoform genes, which are induced by neural activity-dependent expression, are combined into the “vesl-1S / 1M gene” and the two splicing The isoform RNA is collectively referred to as “vesl-1S / 1M”, and the proteins encoded by these two splicing isoform genes or RNA are collectively referred to as “Vesl-1S / 1M protein” or “Vesl-1S / Sometimes referred to as “1M”.

  Vesl-1S / 1M / 1L proteins share the N-terminal 175 amino acids. This N-terminal binds directly to type 1 metabotropic glutamate receptor (mGluR1 / 5), inositol triphosphate receptor (IP3R), ryanodine receptor (RyR), and TRP channel that regulate intracellular calcium concentration, The activity of these receptors is regulated (see, for example, Non-Patent Document 4). In addition, it is linked to NMDA receptor (NMDAR) through binding to Shank to control the activity of NMDAR and AMPA receptor (for example, see Non-Patent Document 5). It is indirectly linked to the actin skeleton system (see, for example, Non-Patent Document 6). Vesl-1L protein has a 191 amino acid region at the C-terminus. There is a coiled-cold structure here, forming a multimer. Vesl-1S protein and Vesl-1M protein lack this C-terminal region and cannot form multimers.

  The most accepted models for the function of Vesl-1 protein are as follows. The constitutively expressed Vesl-1L protein binds to mGluR1 / 5, NMDAR complex, IP3R, RyR, TRP channel, and actin skeletal system. It functions as a scaffold protein that can be stably placed in the body. When neural activity that causes synaptic plasticity occurs, Vesl-1S and Vesl-1M proteins lacking the ability to form multimers are induced, and receptors and channel proteins are formed from the molecular complex formed by Vesl-1L protein. Take away. As a result, it induces rearrangement of functional molecules at the back of the synapse, causing long-lasting synaptic plasticity.

  In this way, Vesl-1S / 1M / 1L regulates glutamate system functions by regulating glutamate receptor activity, regulating intracellular calcium concentration, and regulating the actin skeletal system through molecular rearrangement at the rear synapse. It is thought to have a role to do. Vesl-2 and Vesl-3, like Vesl-1L, have a coiled-coil domain at the C-terminus. Although the expression distribution is different from Vesl-1L, at present, the behavior in the molecule is considered to be the same as Vesl-1L.

  Glutamate is the main excitatory transmitter of the central nervous system and causes many diseases when abnormalities in glutamate function occur. Examples include schizophrenia, affective disorder, alcoholism, epilepsy, learning and memory disorder (for example, see Non-Patent Document 7). Presuming that the Vesl protein family is involved in the regulation of glutamate function, it has been considered that it may be a causative gene of these diseases.

  It has been suggested that one of the causes of schizophrenia is a malfunction of glutamate neurons (see, for example, Non-Patent Document 7). In human schizophrenic patients, two in the vesl-1 intron, two in the vesl-2 intron, one in the 3 'UTR, two synonymous substitutions but two in the vesl-3 translation region SNP has been found. Although a vesl-1 intron SNP was found in an analysis using 680 schizophrenia patients, it is not considered statistically a causative gene for schizophrenia. However, analysis of vesl-1 null knockout mice shows a schizophrenia-like phenotype such as inhibition of pre-pulse inhibition (see, for example, Non-Patent Document 8). Lysergic acid diethylamide (LSD) is a hallucinogenic agent that induces a phenotype similar to symptoms of mental disorders such as acute schizophrenia in humans, but LSD induces the expression of the vesl-1M gene in the frontal cortex (For example, see Non-Patent Document 9). Thus, the vesl-1S / 1M / 1L gene may be a causative gene for symptoms associated with schizophrenia.

  Reward / drug dependence is thought to be mainly related to dopamine system overactivity that projects from the dopamine neurons of the ventral tegmental area to the nucleus accumbens and amygdala. When the dopamine system is activated by administration of the dependent drugs such as cocaine and nicotine, the gene expression level of vesl-1S and vesl-1M increases in the projection target neurons. On the other hand, when the continuous administration of cocaine is stopped, the expression level of Vesl-1L protein decreases (for example, see Non-Patent Document 10). Vesl-1 and vesl-2 null knockout mice that do not express all Vesl-1 or Vesl-2 splicing isoforms are more prominent in hyperactivity induced by cocaine than wild type mice ( Hereinafter, the knockout may be referred to as “KO” and the null knockout may be referred to as “null KO”). In addition, vesl-2 null KO mice are similar to the phenotype of cocaine-addicted animals, such as faster acquisition of cocaine self-administration tasks. Therefore, it is considered that the Vesl protein regulates the activity of the dopamine system through changing the function of the glutamate system (see, for example, Non-Patent Document 11).

It has been reported that even when the dopamine system is activated by the administration of the psychotropic drug methylphenidate, which is used as a therapeutic agent for attention deficit hyperactivity disorder, the expression of vesl-1S gene is increased in the target neurons (for example, non (See Patent Document 12).
Thus, it has been suggested that the vesl-1S, vesl-1M, vesl-1L, and vesl-2 genes are involved in dopamine-related diseases such as reward / drug dependence and hyperactivity.

  Since the vesl-1S gene was isolated as a gene induced by kainic acid convulsions or hippocampal LTP stimulation, it has an important role in the mechanism of convulsions / epilepsy and hippocampal-dependent learning / memory. Was expected. In fact, analysis of mice overexpressing Vesl-1S protein suggests the possibility that Vesl-1S protein has anticonvulsant and antiepileptic effects (see, for example, Non-Patent Document 13). Moreover, vesl-1 null KO mice have poor results in spatial learning ability in the water maze test, which is hippocampal-dependent learning (see, for example, Non-Patent Document 14). In mice where vesl-1S was transiently overexpressed with virus, the memory performance of the object recognition test, a test of hippocampus-dependent memory, was poor. These results suggest that the Vesl-1 isoform may have some important effects on hippocampal-dependent learning memory.

  Vesl family proteins are likely to be good target molecules for the treatment of schizophrenia, affective disorders, reward / drug addiction, epilepsy, and decreased learning and memory in Alzheimer's disease. Therefore, it is necessary to know in detail the functions of each of the splicing isoforms of vesl, and for this purpose, creation and analysis of gene-deficient animals specific to each isoform are desired. To date, gene-deficient mice of the vesl family have been created, but none are null mice that do not express all splicing isoforms of each gene. In particular, vesl-1S / 1M exhibits neuronal activity induction and induces a large amount of expression in the hippocampus, so vesl-1 that has been conventionally produced to know the function of the Vesl-1 protein in the pathogenesis of the disease -1 null KO mice were not analyzed but vesl-1S / 1M KO mice should be used to examine the functions specific to the splicing isoform, and the creation of vesl-1S / 1M-specific KO mice was desired.

In a gene having an alternative 5 ′ splicing donor site in an exon shared between splicing isoforms, there is an isoform that does not undergo splicing at the donor site (FIG. 1A) and an isoform that receives it (FIG. 1B) There is. In addition, in a gene having an alternative 3 ′ splicing acceptor site in an exon shared between splicing isoforms, an isoform (FIG. 1D) that receives and receives an isoform that is not spliced at the acceptor site (FIG. 1D). May exist. For such genes that contain an exon with an alternative 5 ′ splicing donor site or an alternative 3 ′ splicing acceptor, KO mice specific for a particular splicing isoform have been generated due to genetic structural problems. There wasn't. As for vesl-1, a vesl-1S / 1M-specific KO mouse has not been generated due to a problem in the gene structure that it contains an exon having an alternative 5 ′ splicing donor site (FIG. 2).
FEBS Letter 412, 183-189, 1997 J. Neurosci., 22, 167-175, 2002 J Biol Chem 37 (11), 23969-23975, 1998 Neuron 21, 717-726, 1998 Eur J Neurosci 18, 811-819, 2003 Current Biology 13, 50-515, 2003 Annu Rev Pharmacol Toxicol 42, 165-179, 2002 Society for Neuroscience Program No.797.8, 2004 Society for Neuroscience Program No. 741.18, 2002 Eur J Neurosci 21, 1145-1154, 2005 Neuron 43, 401-413, 2004 Neuroscience 132, 855-865, 2005 Eur J Neurosci 16, 2157-2165, 2002 Society for Neuroscience Program No.964.16, 2003

  An object of the present invention is to provide a non-human animal in which the expression level of the vesl-1S / 1M gene has disappeared or decreased compared to the wild type and a method for producing the same. Another object of the present invention is to provide a method for producing a knockout non-human animal specific for a specific splicing isoform for a gene containing an exon having an alternative 5 'splicing donor site or an alternative 3' splicing acceptor.

  As a result of intensive studies in view of the above problems, the present inventors have determined that vesl-1S / is due to deletion of an exon region specific to the vesl-1S gene and inhibition of splicing to the exon specific to the vesl-1M gene. Specifically, by disrupting the 1M gene, a DNA containing exons 7 to 11 unique to the vesl-1L gene is introduced into an alternative 5 ′ splicing donor site existing in the fifth exon of the vesl-1 gene. Constructing a targeting vector for this, introducing it into ES cells to cause homologous recombination, and mating chimeric mice produced with the ES cells, the expression level of the vesl-1S / 1M gene disappears or decreases I found out that I could make a mouse. In addition, vesl-1 null KO mice have a decline in basal motor function, whereas the produced vesl-1S / 1M-specific KO mice have no observed abnormality in basal motor function, but at least abnormal memory retention, It was found to have a phenotype of either abnormal memory refixation, reduced motor learning ability, or increased convulsive resilience. Further, in an exon having an alternative 5 ′ splicing donor site or an alternative 3 ′ splicing acceptor site, a splicing isoform that is subjected to alternative 5 ′ splicing or alternative 3 ′ splicing by deleting a specific region of the exon. Includes exons with alternative 5 'splicing donor sites or alternative 3' splicing acceptors by introducing into the deleted exon region DNA containing all exons necessary for the form to be successfully translated For genes, we found that knockout non-human animals specific for a particular splicing isoform can be generated. 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 the vesl-1S / 1M gene has disappeared or decreased compared to the wild type, or a progeny thereof,
2. 2. The animal according to item 1 above, wherein the disappearance of the expression level of the vesl-1S / 1M gene is caused by disruption of the vesl-1S / 1M gene at two vesl-1 loci on the homologous chromosome.
3. 2. The animal according to item 1, wherein the decrease in the expression level of the vesl-1S / 1M gene is due to the destruction of the vesl-1S / 1M gene at one vesl-1 locus on the homologous chromosome.
4). 4. The animal according to item 2 or 3, wherein the disruption of the vesl-1S / 1M gene is caused by deletion of an exon region specific to the vesl-1S gene and inhibition of splicing to an exon specific to the vesl-1M gene;
5. The animal according to any one of the preceding items 1 to 4, wherein the animal has at least a phenotype of memory retention abnormality, memory re-fixation abnormality, decreased motor learning ability, or increased spasm recovery ability. Animals,
6). The animal according to any one of 1 to 5 above, wherein the animal is a rodent animal,
7). The animal according to item 6 above, wherein the rodent animal is a mouse;
8). A test substance is added to the animal according to any one of items 1 to 7 or a biological sample obtained from the animal, and the influence of the test substance on the animal or the biological sample is detected. A method for screening a therapeutic and / or prophylactic agent for a disease involving 1S protein and / or Vesl-1M protein,
9. 9. The method according to item 8 above, wherein the disease involving Vesl-1S protein and / or Vesl-1M protein is drug dependence, mental disease or neurodegenerative disease;
10. 10. The method according to item 9 above, wherein the drug dependence is due to cocaine or LSD,
11. 10. The method according to item 9 above, wherein the mental illness is schizophrenia.
12 10. The method according to item 9 above, wherein the neurodegenerative disease is Alzheimer's disease.
13. A method for producing a non-human animal in which the expression level of a splicing isoform that does not undergo alternative splicing at an alternative 5 ′ splicing donor site is lost or reduced compared to the wild type, comprising the following steps: Featured method;
(1) a step of deleting a specific exon region downstream from an alternative 5 ′ splicing donor site in an exon having an alternative 5 ′ splicing donor site;
(2) introducing a DNA containing all exons necessary for normal translation of a splicing isoform undergoing alternative 5 ′ splicing into the exon region deleted in (1),
14 A method for producing a non-human animal in which the expression level of a splicing isoform that does not undergo alternative splicing at an alternative 3 ′ splicing acceptor site is lost or reduced as compared to the wild type, comprising the following steps: A method characterized by:
(1) a step of deleting a specific exon region upstream of the alternative 3 ′ splicing acceptor site in the exon having an alternative 3 ′ splicing acceptor site;
(2) introducing a DNA containing all exons necessary for normal translation of a splicing isoform undergoing alternative 3 ′ splicing into the exon region deleted in (1),
15. 14. The method according to item 13 above, wherein the splicing isoform that does not undergo alternative splicing at the alternative 5 ′ splicing donor site is vesl-1S, and the splicing isoform that undergoes alternative 5 ′ splicing is vesl-1L.
Is provided.

  The non-human animal in which the expression level of the vesl-1S / 1M gene of the present invention has disappeared or decreased compared to the wild type is a drug dependence, psychiatric disorder or Vesl-1S protein and / or Vesl-1M protein. It is useful as a model animal for diseases such as neurodegenerative diseases. If the animal is used, it is possible to screen for a therapeutic and / or prophylactic agent for the disease.

The present invention will be described in detail below, but these descriptions are examples (representative examples) of the embodiments of the present invention and do not limit the scope of the present invention. In the present specification, molecular biological techniques such as DNA preparation and vector preparation, protein chemical techniques such as protein extraction and Western blotting, and operations for producing knockout animals are not particularly specified. As long as, Molecular Cloning, A Laboratory Manual, 3 rd edition (Sambrook and Russell, Cold Spring Harbor Laboratory (2001)), Manipulating the Mouse Embryo. A Laboratory Manual, 3 rd edition (Cold Spring Harbor Laboratory, 2003), Gene targeting ( Aizawa, Shinichi; Yodosha (1995)) and the like.

(1) Non-human animal in which the expression level of vesl-1S / 1M gene has disappeared or decreased compared to the wild type The expression level of vesl-1S / 1M gene of the present invention has disappeared or decreased compared to the wild type The non-human animal may be any animal as long as the expression level of the vesl-1S / 1M gene in the animal disappears or decreases compared to the wild type. Methods for controlling the expression level of vesl-1S / 1M gene include, for example, a method of disrupting vesl-1S / 1M gene at two or one vesl-1 gene locus on the homologous chromosome of the animal, Examples include an antisense DNA method, an RNA interference method (Fire A., et al., Nature, 391, 806-811 (1998)), a method of selecting a natural mutant, and the like. In addition, an animal in which the expression of vesl-1S / 1M gene is substantially lost or reduced due to mutation of other genes is also included in the non-human animal of the present invention. In the non-human animal of the present invention, the expression level of the vesl-1L gene is preferably equivalent to that of the wild type.

  In order to produce a non-human animal in which expression of vesl-1S / 1M gene has been lost due to disruption of vesl-1S / 1M gene, both vesl-1 loci of the two vesl-1 loci on the homologous chromosome of the animal are produced. One gene disruption method is used. In order to produce a non-human animal in which the expression of the vesl-1S / 1M gene is reduced, a method of destroying one of the vesl-1S gene loci from the vesl-1 locus of the animal is used. A non-human animal in which one of the vesl-1 loci is disrupted is referred to as a "heterotype animal", and a non-human animal in which both vesl-1S / 1M genes are disrupted. Hereinafter referred to as “homotype animals”. Here, that the vesl-1S / 1M gene is disrupted includes, for example, the introduction of some artificial mutation. Specific examples of disruption of the vesl-1S / 1M gene by artificial mutation include the replacement of all of the vesl-1S / 1M gene with DNA having other nucleotide sequences such as drug resistance genes on the genome. Or, deletion of exon region specific to vesl-1S / 1M gene by deletion of exon region specific to vesl-1S gene and splicing inhibition to exon specific to vesl-1M gene and deletion exon Examples thereof include a method of introducing DNA containing all exons necessary for normal translation of the vesl-1L gene into the region. Specifically, for example, splicing of the fifth exon of vesl-1M by deleting the region corresponding to the C-terminal 11 amino acids of the exon that is specific to the vesl-1S gene in the fifth exon 5th spliced to the 6th exon specific to the vesl-1M gene by destroying the donor site (at the same time, splicing to the 7th exon specific to the vesl-1L gene) A cDNA corresponding to the seventh to eleventh exons may be introduced into the exon region.

  The vesl-1S / 1M gene of the present invention means DNA that comprises all genetic information related to the Vesl-1S / 1M protein, specifically, a coding region that is translated into amino acids, and the region on the genome. A DNA comprising an intron present therein and an untranslated region containing a transcription initiation site 5 ′ upstream or 3 ′ downstream of the coding region. This vesl-1S / 1M gene is, for example, Kato et al., J. Biol. Chem., 273, 23969-23975, (1998), Xiao et al., Neuron, 21, 707-716, (1998), Berke et al., J. Neurosci., 18, 5301-5310, (1998). Examples include polymerase chain reaction (PCR) and part of the vesl-1S / 1M gene from genomic DNA libraries. Can be obtained by hybridization using the above sequence as a probe. It can also be obtained by isolation and extraction from an animal having a vesl-1S / 1M gene by a method usually used. In the present invention, the expression of the vesl-1S / 1M gene refers to the production of mRNA encoding the Vesl-1S / 1M protein and the production of the Vesl-1S / 1M protein.

  As the non-human animal, any animal other than a human having the vesl-1S / 1M gene can be used, but a non-human mammal is preferable. Non-human mammals include, for example, cows, pigs, sheep, goats, monkeys, dogs, cats, rabbits, guinea pigs, hamsters, mice, rats, etc. Among them, ontogeny and biological cycle are relatively short. Also preferred are rodents, especially mice, which are easy to reproduce. Examples of the mouse to be used include C57BL / 6, BALB / c, DBA2, etc. as pure strains, and B6C3F1, BDF1, B6D2F1 strain, ICR, etc. as hybrids, among which C57BL / 6 A system is preferably used.

(2) Production of non-human animals in which the expression level of vesl-1S / 1M gene has disappeared or decreased compared to the wild type expression level of vesl-1S / 1M gene described in (1) above Of non-human animals that have disappeared or declined due to the disruption of the vesl-1S / 1M gene (hereinafter sometimes referred to as “vesl-1S / 1M knockout animals”) Explained. The animal can be prepared using, for example, non-human animal ES cells (embryonic stem cells) in which the vesl-1S / 1M gene is disrupted.

  The vesl-1S / 1M gene of ES cells can be destroyed by the method described above. Specifically, a targeting vector for substituting part or all of the vesl-1S / 1M gene with DNA having another sequence such as a drug resistance gene on the genome is constructed and introduced into the ES cell. Then, it can be performed by selecting cells in which homologous recombination has occurred and the vesl-1S / 1M gene has been disrupted.

(3) Preparation of targeting vector
The targeting vector of the vesl-1S / 1M gene is intended to delete part or all of the vesl-1S / 1M gene. When a part of the vesl-1S / 1M gene is deleted, the vesl-1S / 1M gene is destroyed because, for example, the codon reading frame is shifted or the promoter or exon is inactivated. Deletion of a part or all of a gene by such a targeting vector is usually performed by replacement with DNA having other sequences by homologous recombination. The DNA to be replaced (hereinafter sometimes referred to as “inserted DNA”) includes drug resistance genes such as neomycin resistance (neo) gene and hygromycin resistance gene, lacZ (β-galactosidase gene) and cat Reporter genes such as (chloramphenicol acetyltransferase gene) are preferred because they can also serve as markers for homologous recombination. Furthermore, complete mRNA synthesis can also be inhibited by substitution with a base sequence that terminates transcription of a gene such as a poly A addition signal.

  As such a targeting vector, a DNA between 5 ′ upstream and 3 ′ downstream of a portion to be deleted in the vesl-1S / 1M gene (hereinafter sometimes referred to as “homologous region”) is used. A DNA having a structure in which an inserted DNA is sandwiched (hereinafter sometimes referred to as a “targeting unit”) inserted into an appropriate cloning vector as necessary is used. Here, the homologous region may be any length as long as it is long enough to induce homologous recombination. Specifically, for example, a region of 0.5 kb or more is preferably used. Further, these DNA sequences do not have to completely coincide with the vesl-1S / 1M gene of the cell to be introduced or a sequence in the vicinity thereof, and those having a homology of 80% or more are preferably used. Such a DNA fragment can be obtained from the vesl-1S / 1M gene using a PCR method or the like. For purposes of deletion of the exon region specific for the vesl-1S gene and inhibition of splicing to the exon specific for the vesl-1M gene, the targeting unit is 5 'of the fifth exon of the vesl-1S gene. It has a structure in which a suitable inserted DNA is sandwiched between DNAs consisting of an intron sequence 5 'upstream and 3' downstream from an exon region specific to the vesl-1S gene of the upstream intron and the fifth exon. Such disruption of the vesl-1S / 1M gene can synthesize an incomplete vesl-1S protein consisting of the first to fourth exons and the fifth exon region upstream of the alternative 5 ′ splicing donor site. Therefore, the DNA containing all exons necessary for normal translation of vesl-1L, a splicing isoform that undergoes alternative 5 ′ splicing, ie, exons 7-11, is deleted as described above. It is preferable to introduce into the exon region. The DNA to be introduced includes all exons necessary for normal translation of vesl-1L, and may be genomic DNA including introns as long as these are normally translated. Specifically, for example, cDNA corresponding to the seventh to eleventh exons may be connected so as to be in-frame immediately after the splicing donor site of the fifth exon of the vesl-1 gene.

  The inserted DNA may be selected from those described above. Specifically, for example, a neo cassette “pKJ2” in which a neomycin resistance gene and a PGKpolyA signal are linked downstream of a phosphoglycerate kinase (PGK) promoter (Boer PH, et al., Biochemical Genetics, 28, 299). -308 (1990)) can be used.

  In addition, in the targeting vector of the present invention, the target homologous recombination has occurred although recombination has occurred by using another targeting marker gene inserted 5 ′ upstream or 3 ′ downstream of the above targeting unit. Non-random cells that have undergone random recombination can be excluded. For example, by inserting a gene involved in nucleic acid synthesis such as thymidine kinase gene as another marker, FIAU added to the medium (1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) ) -5-induracil), nucleic acid synthesis inhibitors such as ganciclovir can be taken up into cells where random homologous recombination has occurred and killed to be excluded. Examples of the thymidine kinase (TK) gene include a herpes simplex virus thymidine kinase gene downstream of the MC1 promoter (Thomas KR and Capecchi MR, Cell, 51, 503-512 (1987)). The MC1-TK cassette “F3” or the like obtained by cloning the ligated fragment into the EcoRI / EcoRV site of pBSIISK (−) can be used.

  Examples of the cloning vector include pBS (manufactured by Stratagene), pBluescript II (manufactured by Stratagene), pBR322 (manufactured by TAKARA), pUC18, and pUC19 (manufactured by TAKARA). In addition, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, vaccinia virus or adenovirus vectors, baculovirus, bovine papilloma virus, viruses from the herpesvirus group, animal viruses such as Epstein-Barr virus, etc. Is used.

(4) Introduction of targeting vector into non-human ES cells and selection of homologous recombinants Next, the targeting vector constructed as described above is introduced into non-human ES cells, but targeting is performed to improve efficiency. It is preferable to introduce the vector after making it linear. The cutting position for making the straight chain may be anywhere as long as it is outside the targeting unit. Examples of the introduction method include an electroporation method (electroporation method), a microinjection method, and the like. Among them, the electroporation method is preferable. As non-human ES cells, those that have already been established and stored may be used, or they may be newly established according to a commonly used production method known per se. As non-human animals used for establishing non-human ES cells, the same animals as those described above can be used. For example, in the case of mice, ES cells such as 129 strain are preferably used, and among these, E14TG2a strain (Hooper M., et al., Nature, 326, 292-295 (1987)) and the like are preferably used.

  Nonhuman ES cells into which the targeting vector thus obtained is 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 introducing a drug resistance gene, the drug used for selection is added to the medium at various concentrations, and The concentration may be determined by examining the concentration at which the 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 selection markers is introduced into ES cells of 129 mouse, for example, 50 to 500 μg / ml in the medium. Culture may be performed by adding a neomycin analog such as G418 (manufactured by GIBCO) and a nucleic acid synthesis inhibitor such as 0.1 to 5 μM FIAU. The neomycin analog may be arbitrarily selected from aminoglycoside antibiotics inactivated by a neomycin resistance gene. As the culture medium, a conventionally known and commonly used medium can be arbitrarily selected and used.

  Next, a sufficient number of colonies were picked up from the non-human ES cells cultured as described above, reseeded, and subjected to Proteinase K treatment, heat treatment, etc. to extract DNA, and genotype analysis And non-human ES cells in which homologous recombination has occurred in the vesl-1 gene (hereinafter sometimes referred to as “homologous recombinants”) can be selected more reliably. Genotype analysis methods include PCR using a primer consisting of a base sequence of an inserted DNA and a primer consisting of a base sequence of a region not deleted by homologous recombination, or a base sequence of a region not deleted on the inserted DNA or by homologous recombination Southern hybridization using a probe consisting of The primer or probe used here is any combination as long as homologous recombination has occurred at the target position on the genome of the ES cell and it can be confirmed that the target DNA fragment has been replaced. Also good. As a specific example, for example, when homologous recombination is performed in which an exon region containing a vesl-1S gene-specific sequence is replaced with a part of vesl-1L cDNA (corresponding to exons 7 to 11) and a neomycin resistance gene. Probes used in the analysis include those consisting of the 5 ′ base sequence of the 5 ′ homologous region, those consisting of a partial base sequence of the neomycin resistance gene, and further 3 ′ bases of the 3 ′ homologous region. The thing which consists of arrangement | sequences etc. are mentioned.

  The homologous recombinants obtained in this way usually have a good growth potential, but they tend to lose their ontogenic potential, and therefore need to be subcultured carefully. Cultivation can be carried out by a commonly used method known per se. For example, in a carbon dioxide incubator in the presence of LIF (1 to 10,000 U / ml) on an appropriate feeder cell such as STO fibroblast ( Preferably, the cells can be cultured by a method such as culturing at about 37 ° C. with 5% carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide, 90% air). At the time of subculture, for example, the cells are made into single cells by treatment with a trypsin / EDTA solution (usually using a 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably about 0.1% trypsin / 1 mM EDTA solution). For example, a method of seeding on newly prepared feeder cells is used. A method and conditions suitable for the ES cell used may be selected as long as the obtained homologous recombinant can stably maintain the ability of ontogeny. Specifically, for example, a 129 mouse can be selected. When the E14TG2a strain of derived ES cells is used, feeder cells are unnecessary and can be performed under the same conditions as the culture before selection. Usually, subculture is preferably performed every 1 to 3 days, but it is desirable to observe the cells each time, and to dispose of the cultured cells when morphologically abnormal cells are observed.

(5) Production of chimeric animals and vesl-1S / 1M knockout animals The homologous recombinants thus obtained are cloned, and the cells are cultured at an appropriate early stage of embryogenesis, for example, an 8-cell stage non-human animal embryo or embryo. It is injected into a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the non-human animal pseudopregnant. Alternatively, a chimeric embryo may be produced using an agglutination method co-cultured with an early embryo and transplanted. The produced animal is a chimeric individual composed of both a cell having a normal vesl-1S / 1M gene and a cell having a disrupted vesl-1S / 1M gene (hereinafter referred to as “chimeric animal”). There are things). From an individual group obtained by mating such a chimeric individual and a normal individual (hereinafter sometimes referred to as “F1”), at any one vesl-1 locus on the homologous chromosome, A heterozygous animal can be obtained by selecting an individual having a genotype in which the -1S / 1M gene is disrupted (hereinafter sometimes referred to as "heterotype"). The thus obtained heterozygous animal is a non-human animal in which the expression level of vesl-1S / 1M gene is lower than that of the wild type, and the individual is usually bred and their offspring To obtain a homozygous animal having a genotype in which the vesl-1S / 1M gene is disrupted at two vesl-1 loci on the homologous chromosome (hereinafter sometimes referred to as “homotype”). it can. Such homozygous animals are non-human animals in which the expression level of vesl-1S / 1M gene has disappeared.

Selection of a heterozygous or homozygous animal can be performed, for example, by analyzing the genotype by Southern blotting or PCR using DNA extracted from the tail of the animal as a template. Primers used for analysis by the PCR method may be any combination as long as the genotype can be distinguished from wild type, heterotype, and homotype. For example, a method comprising 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, etc. It is done.
As a specific example, when homologous recombination is performed in which the region containing the vesl-1S gene-specific sequence is replaced with part of the vesl-1L cDNA (exon 7 to 11) and the neomycin resistance gene, the base of the deleted region Sense primer consisting of sequence, antisense primer consisting of base sequence in 3'-side homologous region, sense primer and antisense primer consisting of partial base sequence of neomycin resistance gene, partial base sequence of poly A sequence of bovine growth hormone Use sense and antisense primers. By performing PCR using DNA extracted from the animal as a template, and detecting the resulting PCR product, homozygous, heterozygous, or wild-type can be distinguished. Specifically, for example, a vesl-1 locus in which homologous recombination has occurred with each combination of the primers shown in SEQ ID NOs: 1 and 2 and SEQ ID NOs: 3 and 4 is detected, and SEQ ID NO: 5 in the sequence listing. By detecting the vesl-1 locus where no homologous recombination has occurred with the combination of the primers shown in FIGS. 6 and 6 and SEQ ID NOs: 7 and 8, the genotype of the non-human animal of the present invention can be analyzed.

  Analysis of the expression of the Vesl-1S / 1M protein in the obtained heterozygous and homozygous animals can be performed by Western blotting or the like. Specifically, for example, a protein extract is prepared from various organs, tissues, cells, etc. of the produced hetero-type and homo-type animals, and the extract is electrophoresed and then transferred to an appropriate membrane. The Vesl-1S / 1M protein transcribed above is detected with anti-Vesl-1S / 1M protein serum or the like. Preparation of a protein extract, electrophoresis, transfer to a membrane, detection of a protein, and the like can be performed according to a commonly used method known per se.

(6) Passage and maintenance of heterozygous and homozygous animals The heterozygous and homozygous animals produced in this way can also be used in animal individuals obtained by mating in which the expression level of the gene disappears or decreases. It is possible to carry out breeding in a normal breeding environment. That is, by crossing females and males of heterozygous animals in which the vesl-1S / 1M gene is disrupted at one vesl-1 locus on the homologous chromosome, vesl-1 at two vesl-1 loci on the homologous chromosome Homozygous and heterozygous animals in which the -1S / 1M gene is disrupted can be passaged. In addition, a system can be stably maintained by crossing a heterozygous animal and a wild-type animal. The offspring thus obtained is also included in the non-human animal of the present invention.

(7) Behavioral analysis of vesl-1S / 1M knockout animals The heterozygous and homozygous animals thus obtained should be further analyzed by analyzing the behavior of vesl-1S / 1M protein in order to analyze the function in vivo. Can do.
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 & Sons, Inc.), animal behavioral function test (biological science, published by medical school, vol. 45, No. 5 (1994)) or the like. Specifically, for example, open field test, light / dark selection test, elevated cross test, fear conditioning test, Morris water maze test, prepulse inhibition test, forced swimming test, crossover balance sensory test, wire hanging strength test, electrical Shock sensitivity test, rota-rod test, etc. Among them, for vesl-1S / 1M knockout animals, the light / dark selection test, elevated cross test, fear conditioning test, prepulse inhibition test, and rota-rod test are preferably used, but analysis should be performed by combining several methods. Is preferred.

  In the behavior analysis methods as described above, conditions are set so that changes in a specific phenotype such as physical ability, learning ability, or emotion can be detected. Therefore, such behavioral analysis is performed on the animals and wild type animals (controls) and the results are compared, and if a significant difference is obtained in a particular test, in a certain phenotype that can be analyzed by that test It can be considered that the non-human animal of the present invention has some change or abnormality. Whether or not the obtained numerical difference is significant can be analyzed by an appropriate index. Specifically, for example, after the t-test method, one-way analysis of variance or two-way analysis of variance, It can be performed by a commonly used statistical analysis method such as a method of analysis by the least significant difference method. Such statistical analysis can be easily performed using statistical analysis software such as StatView J (manufactured by Abacus Concepts).

Among the behavioral abnormalities of the non-human animal of the present invention, “abnormality of memory retention” generally means that forgetting of newly learned and memorized matters is faster than control. Such phenotypes include, for example, those in which a decrease in freezing reaction is significantly faster than that of a control individual in a fear conditioning test.
The “abnormality of memory re-fixation” generally means that a process in which a memory once formed becomes stronger as a result of recall is lower than control. As such a phenotype, for example, in the fear conditioning test, after being conditioned once and fear memory is formed, the freezing response after recalling fear memory is significantly lower than that of the control individual And the like.

The “abnormality of motor learning ability” generally means that the ability of the body movement to adapt to the surrounding situation decreases as compared with the control by repetition. Examples of such phenotypes include those in which the delay of the fall time from the rotating rod is significantly lower than that of the control individual in the rota-rod test.
“Increased spasm recovery” generally means that the time required to return to normal behavior such as walking and grooming from generalized convulsions, including limb stiffness, caused by convulsions-induced treatment is shorter than control. Means. As such a phenotype, for example, the time to return to normal behavior after electrical shock is induced by electrostimulation (maximum electro-convulsive seizure, MECS) is significantly higher than that of the control individual. The thing etc. which are shortened are mentioned.

(8) Screening for therapeutic and / or preventive drugs for diseases involving Vesl-1S protein and / or Vesl-1M protein The non-human animal of the present invention has a higher expression level of vesl-1S / 1M gene than wild type Since it has disappeared or decreased, it can be used as a model of a disease caused by the loss of various biological activities generated in the living body by the Vesl-1S protein and / or Vesl-1M protein. Useful for studying methods. That is, the non-human animal of the present invention can be used for screening for therapeutic and / or prophylactic agents for diseases involving Vesl-1S protein and / or Vesl-1M protein. Examples of such diseases include drug addiction, psychiatric diseases or neurodegenerative diseases, and specifically include drug addiction due to cocaine or LSD, schizophrenia, Alzheimer's disease and the like.

  Furthermore, since the non-human animal of the present invention has at least any of the phenotypes of abnormal memory retention, abnormal memory re-fixation, decreased motor learning ability, or increased convulsive resilience, It can be used for screening for therapeutic and / or prophylactic drugs. Specifically, for example, one of the main symptoms of Alzheimer's disease is memory impairment. Since vesl-1S / 1M KO mice exhibit abnormal memory retention, the vesl-1S / 1M gene may be involved in memory impairment in Alzheimer's disease.

  Cocaine administration induces vesl-1S / 1M gene expression. In vesl-1 null KO mice, hyperactivity induced by cocaine is more prominent than wild type. That is, vesl-1 null KO mice show a phenotype similar to that of cocaine poisoned animals. Considering the above two points, it is considered that the vesl-1S / 1M gene whose expression is induced by cocaine administration has a function of suppressing the toxicity (and dependence) to cocaine. In addition, the expression of vesl-1M gene in the frontal cortex by LSD administration suggests that vesl-1M is involved in LSD dependence.

  In addition, vesl-1 null KO mice exhibit schizophrenia-like symptoms such as prepulse inhibition (PPI) inhibition. PPI decline is also seen in human schizophrenic patients and is one of the important criteria for animal models of schizophrenia. These facts suggest that the vesl-1S / 1M KO mouse may be a mouse model of schizophrenia.

  Furthermore, an animal in which another gene is knocked out in the non-human animal of the present invention can be obtained and used 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 expression of another specific gene is lost or reduced. Specific genes include, for example, the mGluR1 / 5 gene, IP3 receptor gene, ryanodine receptor gene, drebrin gene, and shank gene that bind to the EVH (Ena / VASP homology) domain of the Vesl-1S / 1M protein. In addition, genes of molecules localized in post-synaptic thickening involving the Vesl-1S / 1M protein can be mentioned. Such a non-human animal in which the expression of a plurality of genes has disappeared or decreased can also be used for screening for preventive and / or therapeutic drugs for the above-mentioned diseases and the like, thus preventing and / or treating the diseases thus obtained. Drugs are also included in the present invention.

  As a screening method, for example, a test substance is added to the non-human animal of the present invention or a biological sample obtained from the animal, and Vesl-1S protein and / or Vesl-1M protein in the animal or biological sample is involved. Changes in phenotype or vesl-1S / 1M gene expression level can be made by comparing with untreated control animals or biological samples. Examples of biological samples include various organs, tissues, cells, extracts thereof, tissue specimens, cultured cells, body fluids such as blood and urine, and the like. Since the non-human animal of the present invention can be suitably used as a model of, for example, cranial nervous system disease, immune system disease, etc., for analysis of such a disease, a brain slice specimen, a brain neuron cell, an immune system cell, etc. Particularly preferably used. Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel substances. It may be a known substance. The addition amount of the test substance can be appropriately selected according to 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. The symptoms of the test animal and the properties of the test substance are exemplified. What is necessary is just to select suitably according to etc. In the case of using a biological sample obtained from the animal, for example, in the case of using cultured cells, it can be carried out by a method of culturing by adding a test substance to the culture solution. Further, for example, when a brain slice specimen is used, it can be performed by a method such as directly contacting a test substance prepared in a solution form.

  Analysis of phenotypic changes involving the Vesl-1S protein and / or Vesl-1M protein can be performed using, for example, behavioral analysis. Specifically, it can be arbitrarily selected from the various behavior analysis methods as described above, but in the non-human animal of the present invention, a light / dark selection test, an elevated cross test, a fear conditioning test, a prepulse, An inhibition test and a rota-rod test are preferably used. It is also preferable to select and use a behavior analysis method that detects 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 results obtained from the untreated control animals were compared and analyzed. It can be determined that the prepared test substance is useful as a therapeutic and / or prophylactic agent for diseases involving Vesl-1S protein and / or Vesl-1M protein.

  Examples of the analysis of other phenotypic changes include measurement of the amount of various biological substances, measurement of the activity of biological substances, and the like when using the extract of tissues or cells of the non-human animal of the present invention. . In the case of cultured cells, tissue specimens, and the like, morphological changes, measurement of growth rate, measurement of intracellular calcium amount, electrophysiological analysis, measurement of biological substance secretion amount and the like can be mentioned. Also for such analysis, determination can be made by comparing the results when the test substance is added and when there is no treatment in the same manner as described above.

  Further, when the vesl-1S / 1M knockout animal is a heterozygous animal, a change in the expression level of the vesl-1S / 1M gene may be analyzed. Such analysis can be performed using a known method such as Northern blotting or Western blotting, for example, when an extract of tissue or cells is used. In the case of a tissue specimen such as a brain slice, analysis can also be performed by an in situ hybridization method, an immunohistochemical staining method, or the like. When these analyzes are performed and a significant difference is 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 It can be determined that Vesl-1S protein and / or Vesl-1M protein is useful as a therapeutic and / or prophylactic agent for diseases involving the same.

  Using the method as described above, between the vesl-1S / 1M 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. If a significant difference is obtained, the selected substance is used to confirm that the obtained significant difference has an effect on the phenotypic change caused by the disappearance of the expression level of vesl-1S / 1M gene. Furthermore, it is preferable to add to wild-type or heterozygous animals, or biological samples obtained from these animals, and to detect the influence of the substance on them. The wild-type or heterozygous animal used for such analysis is preferably a litter.

(9) Vesl-1S protein and / or Vesl-1M protein therapeutic and / or prophylactic agent for diseases involving Vesl-1S protein and / or Vesl-1M protein A substance determined to be useful as a therapeutic and / or prophylactic agent for a disease can be used as a safe and low-toxic prophylactic and / or therapeutic agent for the disease. A compound derived from a substance obtained by the screening method can also be used in the same manner.

  These compounds may form salts, and may be hydrates and solvates. Examples of the salt of the compound include pharmaceutically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Etc. 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, aluminum salt and ammonium salt. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid. And the like. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ortinine and the like, and preferable examples with acidic amino acid include salts with aspartic acid, glutamic acid and the like.

(10) Method for formulating a therapeutic and / or prophylactic agent for a disease involving Vesl-1S protein and / or Vesl-1M protein The substance described in (9) above alone is used in patients with the above-mentioned diseases, etc. Although these can be administered, one or more of these active ingredients can be mixed and administered. Further, it is preferable to formulate the substance as a pharmaceutical composition using a pharmacologically acceptable additive for pharmaceutical preparation and the like and administer it. For example, tablets, capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublinguals, liquids, elixirs, emulsions, suspensions, etc. As an injection, a suppository, an ointment, a patch or the like manufactured orally as a sterile aqueous liquid or oily liquid. These are, for example, admixed with the physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in unit dosage forms required for accepted pharmaceutical practice. , And can be produced using methods well known in the art such as filling or tableting. The amount of active ingredient in these pharmaceutical compositions is such that an appropriate volume within the indicated range is obtained.

  For example, additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, fillers such as cellulose, mannitol, and lactose. , Starch, polyvinylpolypyrrolidone, starch derivatives, disintegrating agents such as sodium starch glycolate, lubricants such as magnesium stearate, wetting agents such as sodium lauryl sulfate, sucrose, lactose Alternatively, sweeteners such as saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. In addition, tablets can be coated with an enteric coating agent or the like as necessary. As for the capsule, a liquid carrier such as fats and oils can be further contained in the additive.

  In addition, for example, a sterile pharmaceutical composition used as an injection should be formulated in accordance with normal pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil and coconut oil, etc. Can do. As an aqueous solution for injection, for example, isotonic solutions (D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as , Alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.), nonionic surfactant (polysorbate 80TM, HCO-50 etc.) etc. can be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The above pharmaceutical composition includes, for example, a buffer (phosphate buffer, sodium acetate buffer, etc.), a soothing agent (benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (human serum albumin, polyethylene, etc.). Glycol, etc.), preservatives (benzyl alcohol, phenol, etc.), and antioxidants may be blended. The aqueous liquid for injection thus prepared is usually produced by dissolving the substance in a medium as described above and sterilizing by filtration, and then filling it into a suitable sterilized vial or ampoule. To enhance stability, the water in the vial may be removed under vacuum after the pharmaceutical composition has been frozen. An oily liquid can be produced in substantially the same manner as in the case of an aqueous liquid, but can be preferably produced by suspending the compound in the medium and then 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, as a preventive or therapeutic agent for the above-mentioned diseases and the like to humans and animals other than humans. 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. Moreover, it is desirable to administer the daily dose in one to several times.

(11) Other uses of the non-human animal of the present invention The non-human animal of the present invention is characterized in that the expression level of vesl-1S / 1M gene disappears or is lower than that of the wild type. Thus, various biological activities generated in the living body are deleted, and can be used for analysis of various functions of the Vesl-1S / 1M protein.
For example, since the Vesl-1S / 1M protein has an important function in the cerebral nervous system, brain slice samples and cultured cerebral nervous system cells obtained from the animal can be used for analysis of cerebral nervous system functions.

(12) Production of knockout non-human animal specific to a specific splicing isoform In the case of a gene having an alternative 5 ′ splicing donor site in an exon shared between splicing isoforms, the above (2) to (6) In the exon having an alternative 5 ′ splicing donor site, the specific exon region downstream from the alternative 5 ′ splicing donor site of the exon is deleted in accordance with the method described in 1. Knockout non-human animals specific for splicing isoforms that do not undergo alternative 5 'splicing by introducing into the deleted exon region DNA containing all exons necessary for the normal translation of the isoform Can be produced. For example, such a gene includes NF-ATc (Immunity, 10, 261-269, 1999), and a knockout non-human animal specific for NF-ATc / A can be produced.
Further, in the case of a gene having a selective 3 ′ splicing acceptor site in an exon shared between splicing isoforms, the selective 3 ′ splicing acceptor site according to the method described in (2) to (6) above. All exons required for the normal translation of a splicing isoform that undergoes alternative 3 ′ splicing and that lacks a specific exon region upstream of the 3 ′ splicing acceptor site of the exon. By introducing into the exon region from which the DNA containing is deleted, a knockout non-human animal specific for the splicing isoform that does not undergo alternative 3 ′ splicing can be produced. For example, such a gene includes transformer (tra) (Cell, 58, 449-459, 1989), and a knockout non-human animal specific to transformer (non-sex-specific product) can be produced.

EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited at all from these Examples.
Example 1: Generation of Vesl-1S / 1M-deficient mice
In order to analyze the effect of Vesl-1S / 1M protein deficiency on neuronal plasticity, we created vesl-1S / 1M gene-deficient mice. Among the three splicing isoforms of vesl-1 gene, vesl-1S (SEQ ID NO: 9), vesl-1M (SEQ ID NO: 10), and vesl-1L (SEQ ID NO: 11), the sequence specific to vesl-1S is There are only 33 bases corresponding to the C-terminal (11 amino acids: base numbers 526 to 558 of SEQ ID NO: 9, and amino acid numbers 176 to 186 of SEQ ID NO: 12) (FIG. 3). In the deletion of only this region, 11 amino acids were deleted, but there was concern that an incomplete Vesl-1S protein consisting of the remaining 175 amino acids could be synthesized. The incomplete Vesl-1S protein still has binding regions with Vesl-1S binding proteins such as mGluR and ryanodine receptor, so that the deletion of only the 11 amino acids at the C-terminal causes loss of the function of the Vesl-1S protein. I was worried that I couldn't. Therefore, 11 amino acids were deleted, and a cDNA corresponding to the C-terminus (exons 7 to 11) of vesl-1L cDNA (base numbers 528 to 1101 of SEQ ID NO: 11) was substituted with the targeting vector shown in FIG. Knocked in (FIG. 4). The targeting vector consists of diphtheria toxin gene, 1.5 kb 3 ′ of the 4th intron of vesl-1 gene, upstream region of the 5th exon splicing donor site, cDNA corresponding to 7th to 11th exons, bovine growth hormone gene It consists of a poly A signal region, a neomycin resistance gene, and 11 kb on the 5 ′ side of the fifth intron of the vesl-1 gene. As a result, vesl-1S mRNA was not synthesized, and it was expected that the knocked-in vesl-1L mRNA would be expressed instead. It was expected that endogenous vesl-1L and vesl-1M genes would not be expressed as mRNA because the splicing donor site was destroyed and splicing was inhibited.

The mouse strain used for the targeting construct is 129sv, and the ES cell is C57BL / 6J.
It was confirmed by Southern blot analysis using genomic DNA prepared from the brain that the obtained homomouse was deficient in the vesl-1S / 1M gene (FIG. 5). In addition, it was confirmed by homologous Northern blotting using RNA prepared from brain and Western blotting that homo mice did not express vesl-1S / 1M gene and Vesl-1S / 1M protein. .

  Is the Vesl-1L protein expressed in homo mice consistent with the expression level, expression distribution, and neural activity-dependent expression characteristics compared to the endogenous Vesl-1L protein expressed in wild-type mice (WT)? I examined whether. Mice that induced convulsions by electrical stimulation were used to study the expression dependent on neural activity. By Western blotting using brain hippocampus and cerebral cortex extracts (Figure 6) and immunohistochemistry using brain slices, homo mouse Vesl-1L protein was compared to WT Vesl-1L protein. There is no significant difference in the expression level in the hippocampus and cerebral cortex, the same expression distribution in the brain, and the homo mouse Vesl-1L protein does not change its expression due to neural activity as well as WT Vesl-1L protein. confirmed.

From the above results, it was determined that the created homo mouse was a Vesl-1S / 1M specific deficient mouse (KO).
KO grew normally and no abnormal physical characteristics (appearance, walking, fur) were found.
Following the global guidelines for conducting behavioral experiments, the genetic background was crossed back to C57BL / 6J. The behavioral studies described below used mice that were backcrossed 6-9 times. When multiple animals were bred in one cage, it was thought that the frequency of fighting behavior in the cage and the outcome of win / loss had a great influence on emotional properties such as fear.

Example 2: contextual fear conditioning test
For the purpose of knowing the role of Vesl-1S / 1M protein in memory formation, we imposed a fear conditioning task on KO and examined the characteristics of fear learning and memory.
The formation of fear memory is an instinctive ability of animals, and is found in organisms ranging from lower animals to higher animals. For this reason, fear conditioning is considered the optimal model for clarifying the memory formation mechanism. Place the mouse in a 17 x 17.5 x 30 cm chamber with wires on the floor and apply an electric shock from the floor. The mouse learns by associating the experienced fear with the feared situation and forms fear memory (hereinafter, learning by electric shock in this chamber may be referred to as a “learning session”). When the mouse is re-entered into the chamber, it recalls fear memory from the chamber, even if no electrical stimulation is received, and expresses fear responses such as freezing (disappearance of all movements except breathing) and increased heart rate. The proportion of time showing freezing reaction was measured by an automatic measurement system and used as an index of fear memory formation.

Example 3: Fear learning test
We investigated whether Vesl-1S / 1M protein is necessary for fear learning. In FIG. 7, black is a wild type (WT) mouse, gray and diagonal lines are KO, the horizontal axis is the time course (minutes), and the vertical axis is the ratio of freezing reaction time. In KO, the increase in the percentage of freezing reaction time while receiving an electric shock is delayed compared to WT (left figure), but the percentage of freezing reaction time in both groups immediately after five electric shocks ( There was no significant difference in freezing reaction time / observation time (right figure). This means that KO is slow to learn but can eventually learn.

Example 4: Test for retention of fear memory
We investigated whether Vesl-1S / 1M protein is required for retention of fear memory (FIG. 8). Black is WT, slanted line is KO, and the vertical axis indicates the percentage of freezing reaction time. After 14 days of the learning session, the animals were returned to the chamber and tested without electric shock. There was no significant difference in the percentage of freezing time between KO and WT (t-test, p <0.97). When tested in another group of animals 32 days later (Day 33), KO showed a significantly lower freezing rate than WT and showed abnormal retention of remote memory (t-test, p <0.01) (Figure 8). These results indicate that abnormalities occur in the memory (remote memory) formation process from 14 to 32 days after learning in KO. This time is a time when the memory moving from the hippocampus to the cerebral cortex is converted into a remote memory that lasts for 2-3 weeks. From the above results, it was clarified that Vesl-1S / 1M protein plays an indispensable role in the process of converting memory into a strong one that lasts for a lifetime.

Example 5: Refixation test of fear memory Once fear memory was formed firmly, it is erased when protein synthesis of the amygdala is inhibited during recall. This means that fear memory becomes unstable with recall and requires new protein synthesis in the same way as the initial memory immobilization in order to be stored again. This process is called “refixation”. Fear memory becomes unstable after being recalled, and then becomes a stronger memory through the process of re-fixation. Inhibition of re-immobilization has attracted attention as it may lead to artificially reducing fear memory in the treatment of trauma and PTSD.

  We investigated whether Vesl-1S / 1M protein is required for memory re-fixation. Two days after the learning session (Day 3), the animals were placed in the chamber for 6 minutes to remind them of fear memory. Then, after returning to the home cage, the animal was placed in the chamber and tested again on Day 15. In FIG. 9, black is WT, diagonal lines are KO, and the vertical axis indicates the percentage of freezing reaction time. On Day 15, KO showed significantly lower freezing compared to WT (t-test. P <0.001) (left figure). For another group, when tested on Day 15 without recalling on Day 3, KO showed a freezing reaction similar to WT (t-test. P <0.97) (right figure). Therefore, it was found that KO impedes the refixation of fear memory following recall.

Example 6: Refixation and Erasure Test of Fear Memory In the experiment of FIG. 10, recall was performed for 1 minute on Day 2 (1 day after the learning session), and testing was performed on Day 3 (2 days after the learning session) (right diagram). The test was performed on Day 3 without recalling the control group (left figure). Black is WT, gray is KO, the horizontal axis is the time course (minutes), and the vertical axis is the percentage of freezing reaction time. The recalled group of KO showed a freezing reaction similar to WT for the first 2 minutes, but showed a significantly lower freezing reaction than WT from 3 minutes to 6 minutes (right figure). In the control group, both the WT and KO transitions in the same manner for 6 minutes (left figure). This result also shows that in KO, fear memory is reduced by recall.
These results indicate that the Vesl-1S / 1M protein is involved in reimmobilization of fear memory. Furthermore, it is possible that the Vesl-1S / 1M protein is involved in intra-session elimination.

Example 7: Neuronal hyperexcitability test
A clip-type electrode was placed between the WT and KO pinna, and electrical stimulation was applied to induce maximum electro-convulsive seizure (MECS). For electrical stimulation, an electrical pulse with a pulse width of 0.5 milliseconds and 60 mA was applied at 100 Hz for 0.1 second. After recovering from limb stiffness, intermittently repeats “rest of limbs” and “rise, grooming, walking”. By observing this behavior, we investigated the involvement of the Vesl-1S / 1M protein in convulsions.

  FIG. 11 shows data for each individual. The horizontal axis shows the time (minutes) after MECS stimulation. On the vertical axis, 0 indicates “rest of limbs” and 1 indicates “non-stationary state such as standing, grooming, walking”. WT moved about 5-15 minutes after MECS stimulation, but remained almost stationary until 60 minutes later. In KO, it moved well until around 20 minutes after MECS stimulation. The moving time was also longer than WT.

Immediately after electrical stimulation inducing MECS, KO mice were more likely to die immediately than WT. In addition, when convulsions were induced by intraperitoneally administering kainate, an excitatory neurotransmitter receptor agonist, KO was more likely to die immediately than WT.
Therefore, it is suggested that KO is vulnerable to convulsions and that the mechanism of recovery from convulsions works more strongly than WT after convulsions occur.

Example 8: Rota-rod test
The Rota-rod test is a method of measuring motor learning ability. In this test, a mouse is placed on a 3 cm diameter rod whose rotation gradually accelerates from 3 rpm to 30 rpm in 300 seconds, and the number of seconds after which it falls is measured (upper figure 12). In the wild-type mouse, even if the rotation speed of the rod is slow in the first trial, the forelimbs and hindlimbs do not move cooperatively and fall off the rod. As the trials are repeated, even if the rotational speed increases to learn the cooperative movement of the forelimbs and hindlimbs, it will not fall easily. This test can test basic motor ability and motor learning ability.

  The basic motor ability and motor learning ability of KO were examined by Rota-rod test. KO was not significantly different from WT in the first and second trials. When the trials were repeated, WT had a longer time to fall, whereas KO had almost the same time as the first trial (bottom of Fig. 12). This suggests that in KO, there is no abnormality in basic motor ability, but there is a defect in motor learning ability.

FIG. 3 is a schematic diagram showing alternative 5 'splicing and alternative 3' splicing. The black portions of A and B correspond to specific exon regions, respectively. FIG. 2 is a schematic diagram showing alternative 5 'splicing of vesl-1. The square frame of vesl-1S represents the fifth exon, and the shaded area corresponds to a specific exon region. The left square frame of vesl-1M represents the fifth exon, the right square frame represents the sixth exon, the left square frame of vesl-1L represents the fifth exon, and the right square frame represents the seventh exon. The first to fourth exons and the eighth to eleventh exons are omitted. It is the schematic diagram which compared the protein structure of Vesl-1 family. FIG. 2 is a schematic diagram showing a targeting vector for producing the vesl-1S / 1M KO mouse of the present invention. DT-A is the diphtheria toxin gene, E5 is the 5th exon, E7-E11 is the cDNA corresponding to the 7th to 11th exons, pA signal is the polyA signal region of bovine growth hormone, promoter-neo ^r -pA Indicates a neomycin resistance gene, respectively. It is a figure which shows the result of the Southern blot analysis of this invention vesl-1S / 1M KO mouse | mouth. M represents a size marker. + / + Represents a wild type mouse, +/− represents a heterozygous mouse, and − / − represents a homozygous mouse. It is a figure which shows the result of having compared the expression level of the Vesl-1L protein which this invention vesl-1S / 1M KO mouse | mouth expresses with a wild type mouse | mouth by Western blotting. + / + Represents a wild type mouse, and − / − represents a homozygous mouse. M represents a size marker and r-1L represents a control recombinant Vesl-1L protein. It is a graph which shows acquisition of fear of the fear conditioning task performed about this invention vesl-1S / 1M KO mouse | mouth. The arrow in the left figure represents the time when the electric shock was given. Moreover, the right figure shows the ratio of the freezing reaction time for 3 minutes immediately after five electric shocks. It is a graph which shows holding | maintenance of the remote memory of the fear conditioning task performed about this invention vesl-1S / 1M KO mouse | mouth. The left figure shows the percentage of the freezing reaction time for 3 minutes immediately after the learning session (5 electric shocks). The right figure shows the ratio of the freezing reaction time for 3 minutes 32 days after the learning session. It is a graph which shows re-immobilization of fear memory of the fear conditioning task performed about this invention vesl-1S / 1M KO mouse | mouth. It is a graph which shows the refixation of fear memory of the fear conditioning task performed about this invention vesl-1S / 1M KO mouse | mouth, and the deletion in a session. It is the result of having observed the action from immediately after inducing convulsions by the electrical stimulation performed about this invention vesl-1S / 1M KO mouse | mouth to 1 hour later. It is a graph which shows the result of the rota-rod test performed about this invention vesl-1S / 1M KO mouse | mouth. Circles indicate WT results and squares indicate KO results.

Claims (15)

A non-human animal in which the expression level of vesl-1S / 1M gene has disappeared or decreased compared to the wild type, or a progeny thereof. The animal according to claim 1, wherein the disappearance of the expression level of the vesl-1S / 1M gene is due to the disruption of the vesl-1S / 1M gene at two vesl-1 loci on the homologous chromosome. The animal according to claim 1, wherein the decrease in the expression level of the vesl-1S / 1M gene is due to the disruption of the vesl-1S / 1M gene at one vesl-1 locus on the homologous chromosome. The animal according to claim 2 or 3, wherein the disruption of the vesl-1S / 1M gene is caused by deletion of an exon region specific to the vesl-1S gene and inhibition of splicing to an exon specific to the vesl-1M gene. . 5. The animal according to any one of claims 1 to 4, wherein the animal has at least a phenotype of abnormal memory retention, abnormal memory re-fixation, decreased motor learning ability, or increased convulsive resilience. The animal described. The animal according to any one of claims 1 to 5, wherein the animal is a rodent animal. The animal according to claim 6, wherein the rodent animal is a mouse. 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, and the influence of the test substance on the animal or the biological sample is detected. A method for screening a therapeutic and / or prophylactic agent for a disease involving -1S protein and / or Vesl-1M protein. The method according to claim 8, wherein the disease involving Vesl-1S protein and / or Vesl-1M protein is drug dependence, psychiatric disease or neurodegenerative disease. 10. The method of claim 9, wherein the drug addiction is due to cocaine or LSD. The method according to claim 9, wherein the mental illness is schizophrenia. The method according to claim 9, wherein the neurodegenerative disease is Alzheimer's disease. A method for producing a non-human animal in which the expression level of a splicing isoform that does not undergo alternative splicing at an alternative 5 ′ splicing donor site is lost or reduced compared to the wild type, comprising the following steps: Featured method;
(1) a step of deleting a specific exon region downstream from an alternative 5 ′ splicing donor site in an exon having an alternative 5 ′ splicing donor site;
(2) A step of introducing DNA containing all exons necessary for normal translation of a splicing isoform undergoing alternative 5 ′ splicing into the exon region deleted in (1). A method for producing a non-human animal in which the expression level of a splicing isoform that does not undergo alternative splicing at an alternative 3 ′ splicing acceptor site is lost or reduced as compared to the wild type, comprising the following steps: A method characterized by:
(1) a step of deleting a specific exon region upstream of the alternative 3 ′ splicing acceptor site in the exon having an alternative 3 ′ splicing acceptor site;
(2) A step of introducing DNA containing all exons necessary for normal translation of a splicing isoform undergoing alternative 3 ′ splicing into the exon region deleted in (1). 14. The method of claim 13, wherein the splicing isoform that does not undergo alternative splicing at the alternative 5 'splicing donor site is vesl-1S and the splicing isoform that undergoes alternative 5' splicing is vesl-1L.