JPH11196709A - Transgenic mouse deficient in doc2 alpha gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transgenic mouse, useful as an experimental animal having excellent synapse response, for elucidating the condition physiology and cause of nervous system and endocrine system in which Doc2α participates, developing a therapy, etc., by making the transgenic mouse deficient in Doc2αgone. SOLUTION: This transgenic mouse is made deficient in Doc2α gene by substitution or insertion of another gene such as a selected marker gene or the like in any partial sequence of Doc2α gene. Preferably the transgenic mouse is prepared by substituting a partial sequence containing a part of a first exon of Doc2α gene with the selected marker gene or inserting the gene into the partial sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a transgenic mouse lacking the Doc2α gene.

[0002]

BACKGROUND OF THE INVENTION The release of neurotransmitters from nerve cells
It is achieved by the fusion of presynaptic membranes with synaptic vesicles that store neurotransmitters. This process is similar to the mechanism by which a physiologically active substance such as a hormone is released outside a cell by exocytosis. The present inventors have discovered human Doc2α as a molecule involved in neurotransmitter release and hormone secretion. Both human and mouse Doc2α are brain-specific proteins with a molecular weight of 45 kDa, and have two C2 regions on the carboxy terminal side (see FIG. 1). The C2 region is known as a region that binds to calcium ions and phosphatidylserine (Orit
a, S. et al., Biochem. Biophys. Res.Commun. 206:
439, 1995). The present inventors have confirmed that Doc2α is specifically expressed in brain nerve cells, and by a cell fractionation method,
Most of Doc2α is present in high concentrations in the synaptic vesicle fraction,
And it was revealed that a part was present in the synaptic membrane fraction (Naito, A. et al., Mol. Brain Res. 44: 198,
1997; and Orita, S. etal., Biochem. Biophys. Re.
s. Commun. 206: 439, 1995). Furthermore, the present inventors have shown that overexpression of Doc2α in PC12 cells promotes the secretion of growth hormone released by a mechanism similar to neurotransmitters (Orita, S. et al.). .,
J. Biol. Chem. 271: 7257, 1996). These results suggest that Doc2α plays an important role in neurotransmitter release.

Recently, it has been shown that Munc18 binds to the C2 region of Doc2α (Verhage, M. et al., Neuron 18: 45).
3. 1997). Separately, the present inventors have found that Doc2
Munc13-1 was found as a protein that binds to the amino terminal region of α (Mid in FIG. 1) (Orita, S. et al., J. Bio).
l. Chem. 272: 16081, 1997). Munc18 and Munc13 are
It is a mammalian homolog of the nematode proteins unc18 and unc13. Both unc18 and unc13 are causative genes of behavioral abnormalities in nematodes, and have been shown to be involved in neurotransmitter release. Therefore, Doc2α is Munc
By binding to 18 and Munc13, it is thought to be working on the release of neurotransmitters.

Thus, to date, attempts have been made to elucidate the physiological function of Doc2α through biochemical analysis of Doc2α. However, it has not been clarified how Doc2α is involved in the physiology of nerve cells in actual living organisms.
Therefore, it is desired to provide an animal model useful for elucidating the physiological function of Doc2α.

[0005]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned problems, and makes it possible to directly elucidate the physiological function of Doc2α, and to improve various pathological conditions involving Doc2α. The purpose of the present invention is to provide an animal model with a clear genetic background, which is useful for elucidating or studying therapeutic methods.

[0006]

Means for Solving the Problems The transgenic mouse of the present invention is a mouse lacking the Doc2α gene.

[0007] In a preferred embodiment, the transgenic mouse of the present invention is a mouse lacking the Doc2α gene by substituting or inserting another gene for any partial sequence of the Doc2α gene. Other genes can be selectable marker genes.

[0008] In a more preferred embodiment, the transgenic mouse of the present invention is a mouse in which a selective marker gene has been substituted or inserted into a partial sequence containing a part of the first exon of the Doc2α gene.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

In the present invention, unless otherwise indicated,
Recombinant DNA methods, gene transfer methods, protein separation and analysis methods, and immunological methods known in the art may be employed. These techniques involve commercially available enzymes, kits, antibodies,
It can be performed using a labeling substance or the like.

(Definition of terms) In this specification, "Doc2
The “α gene” includes a mouse gene having the cDNA sequence shown in SEQ ID NO: 1 in the sequence listing, a gene having a genomic sequence corresponding thereto, a natural mutant thereof, an allelic mutant thereof, and the like. The Doc2α gene includes from the transcription regulatory region to the translation stop codon, the primary transcript of which consists of 10 exons and 9 introns (FIG. 2A). In the genomic DNA corresponding to the cDNA sequence shown in SEQ ID NO: 1, between nucleotides 637 and 638, 717 and 7
Between 18th, between 792 and 793, between 902 and 903, between 1029 and 1030, between 1089 and 1090, 12
Between positions 53 and 1254, between positions 1335 and 1336, and 14
There is an intron between positions 32 and 1433.

As used herein, the term "transgenic mouse deficient in the Doc2α gene" refers to a mouse into which a gene has been introduced by genetic manipulations well known in the art. When performing Southern blotting using the full length as a probe, when compared to Southern blots on DNA from wild-type mice,
A mouse showing a difference equal to or more than a detectable level. This transgenic mouse may be deficient in the Doc2α gene heterozygously or homozygously, but is preferably homozygously deficient.

In a mouse in which the Doc2α gene is homozygously deleted, it is preferable that a 45-kDa Doc2α protein derived from a wild-type mouse cannot be detected when an arbitrary antibody against the Doc2α protein is used. More preferably, there is no detectable protein when used. These detections are performed, for example, by the anti-Doc2α antibodies described herein.

As used herein, “any partial sequence of the Doc2α gene” refers to any partial sequence from the transcription regulatory region of the Doc2α gene to the translation stop codon. This subsequence preferably comprises part of an exon, more preferably part of the first exon. Hereinafter, this “partial sequence” is also referred to as “target sequence”.

As used herein, “other genes”
Refers to a nucleotide having a sequence other than the sequence of the Doc2α gene. Other genes may or may not encode a protein. Preferably, the other gene encodes a protein, and more preferably encodes a selectable marker.

(Preparation of Mouse Deficient in Doc2α Gene)
The transgenic mouse of the present invention can be obtained by deleting the Doc2α gene. As a method for deleting the Doc2α gene, typically, gene targeting by homologous recombination of the gene, Cre / loxP recombination system (Ku
hn, R. et al., Science 269: 1427-1429 (1995)).

The principle of gene targeting is known, and the target gene to be deleted is specifically deleted by homologous recombination. In homologous recombination, DNA having a modified sequence is introduced into a cell to replace a partial sequence in a target gene (that is, a target sequence). Here, the DNA to be introduced includes a modified sequence and sequences at both ends thereof that are homologous to the corresponding both ends in the target sequence (hereinafter, referred to as homologous sequences). Therefore, the above DNA
Has a sequence that is different from the corresponding part in the target sequence in the modified sequence. In the cell, homologous recombination occurs between the cell's native target gene and the introduced DNA, resulting in the replacement of the cell's target sequence with the modified sequence. In this way, the target gene is modified.

In gene targeting for deleting the Doc2α gene, the target sequence may be a part of the Doc2α gene or the whole. The target sequence may be a sequence present in an exon or an intron of the Doc2α genomic DNA, or may be a sequence present in a regulatory region of the Doc2α gene. The target sequence is preferably a sequence containing a part of the exon of the genomic DNA of the Doc2α gene, and more preferably a sequence containing a part of the first exon of the Doc2α gene. The first exon of the Doc2α gene encodes an initiation codon as well as amino acids in the N-terminal region (FIG. 2). The length of the target sequence is not particularly limited, and can be arbitrarily set by those skilled in the art.

The sequence corresponding to the target sequence is a DNA encoding mouse Doc2α which has been cloned, for example, a mouse Doc2α cDNA (Naito, A. et al., Mol. B
rainRes. 44: 198, 1997) and screening a genomic DNA library of mouse liver.

Once the sequence corresponding to the target sequence has been obtained, the middle part of this sequence is modified. Such modification can be performed by a gene recombination method well known in the art (Maniatis, T. et al., Molecul).
ar Cloning: A Laboratory Manual, 2nd edition, Cold Spri
ng Harbor Laboratory, New York (1989)). Sequence modification includes deletion, insertion,
And substitutions. If the sequence modification is an insertion,
The target sequence may consist essentially of only the sequence corresponding to the homologous sequence described above. Modification by substitution or insertion is preferred. The modification needs to be sufficient to delete the Doc2α gene. The sequence used for insertion and substitution can be any sequence, but is preferably a sequence encoding a gene. Examples of genes used for insertion and replacement include a selectable marker gene. Use of a selectable marker gene facilitates detection of the desired alteration. Examples of selectable marker genes include a neomycin resistance gene (selected by G418 (Geneticin) resistance), a β-lactamase gene, a diphtheria toxin A fragment gene,
Examples include the herpes virus thymidine kinase gene and the hygromycin resistance gene. Modifications using these sequences can be performed in vitro by conventional DNA recombination methods.

A DNA sequence having a modified sequence can be incorporated into an appropriate plasmid, vector, or the like and amplified in a large amount.

The DNA for homologous recombination thus obtained
Is introduced into totipotent cells. Examples of totipotent cells include mouse embryonic stem cells (ES cells) and embryonic tumor cells (EC cells). Examples of a method for introducing DNA for homologous recombination include an electroporation method, a microinjection method, a DEAE-dextran method, and a calcium phosphate method. As described above, in cells into which DNA for homologous recombination has been introduced,
Homologous recombination occurs between the DNA (target sequence) of the 2α gene and the introduced DNA, and the target sequence is replaced with a modified sequence in the introduced DNA. If the modified sequence has a selectable marker gene, this marker gene is Doc2α
Introduced into the gene.

Next, cells in which homologous recombination has occurred are selected. Selection can be performed, for example, by detecting the phenotype of the selectable marker, hybridization with the target sequence by Southern blotting, or analyzing the length of the amplified fragment by PCR. The use of selection markers
This is preferable in that this selection is facilitated.

The cells in which homologous recombination has occurred are injected into an early mouse embryo. The stages of early embryo development include blastocysts, 8
Cell-stage embryos and the like. Blastocysts are preferred.

Next, the embryos after injection are transplanted into the uterine horn of pseudopregnant mice to obtain offspring. The offspring are
Become a chimeric mouse. Litters are obtained by crossing the obtained chimeric mice with other appropriate strains of mice. Germ cells of chimeric mice are homologous recombinants (ie, Doc2
A mouse in which one of the Doc2α genes has been modified (a heterozygote; also referred to as a heterozygous mutant mouse) can be obtained if it is derived from a cell lacking the α gene. Homozygotes that do not have a normal Doc2α gene by crossing heterozygous mice (also called homozygous mutant mice)
Transgenic mice can be obtained. The transgenic mouse of the present invention refers to a homozygous and heterozygous transgenic mouse.

Using the obtained hetero or homozygous transgenic mouse, the pathophysiology and causes of nervous system / endocrine system diseases involving Doc2α protein can be elucidated, and therapeutic methods can be developed. For example, if a histological study is performed on a transgenic mouse lacking the Doc2α gene, and a pathological condition that is not seen in a non-transgenic animal is found, this pathological condition is associated with the absence of Doc2α expression. For example, to examine the role of the Doc2α protein in neurotransmission, long-term enhancement in the hippocampus of transgenic and non-transgenic mice can be measured and compared. In order to develop a therapeutic method for a disease associated with the Doc2α protein, a candidate treatment can be administered to the transgenic mouse of the present invention to evaluate the results. The development of such a therapy using model animals is well known in the art, and can be performed by those skilled in the art by setting appropriate conditions. For example, for the development of a drug to be used for the treatment of a disease involving the Doc2α protein, it is useful to administer a candidate drug to the transgenic mouse of the present invention and observe whether the pathological condition has been improved. Drug can be determined.

[0027]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

Example 1 Preparation of Construct for Homologous Recombination of Mouse Doc2α Gene Genomic DNA of 129SVJ mouse prepared according to a conventional method was
Amplified by CR and contains about 15 codons for the mouse Doc2α start codon.
The DNA sequence of 00b was obtained. The sequences of the two oligonucleotides used as PCR primers are shown in SEQ ID NOs: 2 and 3 in the sequence listing. A 129SVJ mouse liver genomic library (obtained from Stratagene) was screened using the above DNA sequence amplified by PCR as a probe. Positive plaques were selected. Using the obtained plaque, a phage was prepared according to a conventional method, and DNA was extracted from the phage. This DNA is converted to the restriction enzyme Sal I
And a DNA fragment of about 20 Kb was recovered. This DNA
The fragment was subcloned using the plasmid pBluescript. The sequence of this subclone was determined. This subclone had a sequence containing the first exon of the mouse Doc2α gene.

Next, a targeting vector was constructed as a construct for homologous recombination of clones (FIG. 2B).
STOP codon downstream from Bgl II site upstream of Doc2α gene
The part up to the HindIII site is used as a target sequence, and the SphI site of the first exon to HindII of the third exon of the Doc2α gene
The part up to the I site was modified. This modification,
To enable positive selection of homologous recombinants,
This was performed by replacing the Bgl II-Hind III part with the β-lactamase gene and the neomycin resistance gene.
To perform a negative selection of homologous recombinants, a diphtheria toxin A fragment was incorporated in a reverse orientation downstream of the HindIII site downstream of the STOP codon. The neomycin resistance gene and the β-lactamase gene were integrated so as to be in-frame. A Bgl II-Sph I portion (3.0 kb, 5 ′ to the translation initiation site) and a Hind III-Hind III portion (5.0 kb, third
The exon (3 'side) is used as a homologous sequence.

The targeting vector and the mouse
When homologous recombination occurs with the Doc2α gene,
The region from the translation initiation point to the third exon (1.0 kb) is β-
Lactamase gene-neomycin resistance gene (5.2kb)
(FIG. 2C). As a result, the β-lactamase gene product and the neomycin resistance gene product are expressed by the mouse Doc2α promoter. Targeting vector is mouse genomic DNA by non-specific recombination
When diphtheria toxin A is incorporated downstream of the vector, such cells are killed because they are expressed in mouse cells. Therefore, cells that survive in the selection medium containing neomycin are cells in which specific homologous recombination has occurred in the Doc2α gene.

Example 2 ES cells and culture method thereof ES cells derived from 129 / SVJ mouse blastocyst E14 strain (Ho
oper, ML et al. Nature 326: 292, 1987). For culture of ES cells, Dulbecco's modified Eagle's medium (D
-MEM, 12800-066, manufactured by GIBCO) and 15% fetal bovine serum (FC
S), 0.1 mM 2-mercaptoethanol, nucleic acid mixture (80
μg / ml adenosine, 85 μg / ml guanosine, 73 μg / ml cytidine, 73 μg / ml uridine, and 24 μg / ml thymidine), 0.1 mM non-essential amino acid solution, 2 mM L-glutamine,
A culture solution (ES medium) containing 50 units / ml penicillin, 50 μg / ml streptomycin, and 10 ³ units / ml LIF (ESGRO: leukemia cell inhibitory factor, manufactured by AMRAD) was used. ES
The cells were passaged at 37 ° C. for 5 minutes in a solution containing 0.25% trypsin and 0.04% EDTA (TE solution, manufactured by GIBCO), and then dispersed into single cells by pipetting.
This was performed by seeding on a feeder cell layer. Culture medium was changed at 12 hour intervals.

For culturing mouse embryonic fibroblasts used as feeder cells for ES cells, 20% FC was added to D-MEM medium.
S, 2 mM L-glutamine, 50 units / ml penicillin, and
A medium (EF medium) supplemented with 50 μg / ml streptomycin was used. Preparation and culture of mouse fetal fibroblasts were performed as follows. Fetuses of C57BL / 6J mice of 10-14 days of gestation were aseptically collected. The fetus was washed with PBS, the head and internal organs were removed, and the remaining part was minced with scissors. Subsequently, the minced sections were treated in a TE solution (0.05% trypsin and 0.53 mM EDTA) at 37 ° C. for 5 minutes to obtain a cell suspension. The resulting cell suspension was filtered through a nylon mesh to remove cell clumps and tissue debris. The filtered cell suspension was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed to obtain a cell precipitate. The cell precipitate was suspended in 30 ml of EF medium and cultured at 37 ° C. in a 175 ml culture flask. Passages were performed at 3-4 day intervals and cells at the third passage were treated with mitomycin C to prevent further cell division. Cells treated with mitomycin C were used as feeder cells. Treatment with mitomycin C was performed as follows: confluent fetal mouse fibroblasts were treated with EF medium containing 10 μg / ml mitomycin C for 2 hours and washed three times with EF medium. And then
The cells were detached from the flask by treatment with a TE solution at 37 ° C. for 5 minutes; the detached cells were centrifuged at 1500 rpm for 5 minutes to obtain cell pellets;
The cells were suspended in S, 10% dimethyl sulfoxide (DMSO) to adjust the cell number to 6 × 10 ⁶ cells / ml, and then frozen and stored in liquid nitrogen. Feeder cells are placed on gelatin-coated flasks, dishes, or microplates.
What was seeded at ４4 × 10 ⁴ cells / cm ² was used.

Example 3 Doc2 of ES cells by homologous recombination
Mutation of α gene DNA for homologous recombination linearized by restriction enzyme Not I
40 μg were suspended in PBS containing 2.3-2.5 × 10 ⁶ E14 strain ES cells. Gene transfer was performed by electroporating the suspension at 800 V and 3 mF. After gene transfer, 125 μg / ml G418 (Genetisin, Sigma
ES cells containing homologous recombination were selected by selective culture at 37 ° C. for 150 hours in an ES medium containing the same. G4
For 18 resistant colonies, about 150 hours after gene transfer, use a micropipette to contain 20 μl of TE solution 96
Microplate with holes (Corning 25850, Corning)
And treated at 37 ° C. for several minutes, then disperse the cells by pipetting, and transfer to a 96-well microplate containing 200 μl of ES cell medium (ES medium described above),
Culture was continued at 37 ° C. When the cells on the 96-well microplate reach confluence, 37 minutes at 37 ° C
After TE treatment, the cells were transferred to a 24-well microplate (IWAKI 3820-024, manufactured by Iwaki Glass Co., Ltd.) containing 3 ml of ES medium, and cultured to grow the cells. The ES cells were all cultured on feeder cells.

The homologous recombinant was confirmed by the Southern blotting method (see Maniatis, T. et al., Supra) as follows. Protease / phenol extraction method from G418 resistant cells (Gomi, H. et al., (1995) Neuron 14, 29-4
Genomic DNA was extracted according to 1). Using two types of restriction enzymes and two types of probes, recombination on the 5 ′ side and 3 ′ side was confirmed. Confirmation on the 3 'side confirmed that genomic DNA was
Digestion with HI and the EcoR I-BamHI fragment shown in FIG. 2A
(0.66 kb 3 ′ probe) was used as a probe. In the homologous recombinant, a 6.6 kb band was detected,
In the wild type, since a band of 8.6 kb is detected, the genotype can be confirmed by the size of the detected band.
Cells confirmed to have a homologous recombinant genome by this method were further confirmed for 5 ′ recombination. For confirmation on the 5 'side, genomic DNA was digested with restriction enzyme EcoR I, and the EcoR I-Bgl II fragment (0.3 kb 5'p
robe) as a probe. In the homologous recombinant, a band of 6.4 kb is detected, and in the wild type, a band of 5.6 kb is detected, so that the genotype can be confirmed by the size of the detected band.

The number of colonies confirmed to be homologous recombinants by confirmation on the 3 ′ side and 5 ′ side was 49 out of 288 G418 resistant colonies.

Example 4 Preparation of Chimeric Mice Using Doc2α Gene Mutant ES Cells ES having Mutations in Doc2α Gene Confirmed with Homologous Recombination
The cells were injected into blastocysts and then transplanted into the uterine horn of pseudopregnant mice to obtain offspring.

Embryos of C57BL / 6J strain mice were collected on day 3.5 of natural mating by perfusing the uterus with PBS containing 5% FCS. 15-20 homologous recombinant ES cells per blastocyst were injected into the collected blastocysts of C57BL / 6J mice as follows.

The ES cells used for injection were prepared as follows. Thaw frozen ES cells 20 hours before injection,
Culture was started on feeder cells at 37 ° C. in ES medium. After dispersing ES cells and feeder cells by TE treatment at 37 ° C. for 5 minutes, the cells were allowed to stand on a gelatin-coated 24-well plate for 30 minutes to remove only the feeder cells.
Only ES cells were collected by attaching to the bottom of a 24-well plate and collecting the cell suspension. The ES cells were kept on ice until subjected to micromanipulation. The injection needle used for injecting ES cells narrows the capillary (MICROCAPS 50 μl),
Among the crushed tips, only those needles whose tips were sharply cut and the inside diameter of the tip of the capillary was slightly smaller than that of ES cells were used. The holding needle used to hold the embryo was produced by cutting the capillary drawn by the above-described method and then melting the tip.

Each of the injection needle and the holding needle has a
After bending 30 degrees, the micromanipulator (NARISI
GE). The chamber used for microscopic operation is 5cm
ES containing 10 mM Hepes buffer using a Petri dish lid
A drop of medium (pH 7.3) was placed and the top was covered with mineral oil. Blastocysts collected from the uterus are at 37 ° C and 5% C
The cells were cultured and expanded in 10 mM Hepes buffered ES medium under O ₂ conditions for 1 hour. The blastocysts and ES cells were put into a drop using a glass needle, and micromanipulated under an inverted microscope. 15-20 homologous recombinant ES cells were injected per blastocyst.

The engineered embryo into which the ES cells have been injected is placed in an ES medium.
After culturing at 37 ° C. for 1 hour, the mice were transplanted into the uterine horn of ICR recipient females (8 mice) on day 2.5 of pseudopregnancy. Of the 30 pups obtained, 23 were determined to be chimeric mice based on coat color. Of these 23 offspring, 13 were male and 10 were female.

Example 5 Preparation of Homozygous Mutant Mice a) Mating of Chimeric Mice Of the 23 chimeric mice obtained by transplantation, 11 having a high contribution of ES cells were crossed with C57BL / 6J mice. If the germ cells of the chimeric mouse are derived from a homologous recombinant, the coat color of the offspring to be delivered will be wild (Agouchi (brown)
Color), and black if derived from blastocysts of C57BL / 6J mice. From the coat color of offspring of 11 chimeric mice, 6 chimeric mice out of 11 chimeric mice were:
It was confirmed that the ES cells were transmitted to the germ line.

From the six chimeric mice confirmed to have transmitted ES cells to the germ line, a total of 164 offspring with wild coat color were obtained by crossing with C57BL / 6J mice. Was.

For these wild-haired mice,
Genotype analysis was performed. Genomic DNA was prepared from the tip of the tail of a 2-3 week old mouse, digested with the restriction enzyme Bam HI, and subjected to Southern blotting using the 3 ′ probe of FIG. It was determined. As a result, it was confirmed that Doc2α gene was heterozygously mutated in 85 out of 164 animals.

B) Mating of heterozygous mutant mice Male heterozygous Doc2α gene mutant mice were backcrossed with female C57BL / 6J strain mice at least once in order to replace ES cell-derived chromosomes. Offspring obtained by backcrossing were subjected to Southern blotting as described in a) above to confirm the mutation of the Doc2α gene. The Doc2α gene heterozygous mutant mice thus obtained were crossed with each other, and the Doc2α gene mutation in the offspring was confirmed by Southern blotting to obtain a homozygous Doc2α gene mutant mouse.

FIG. 3 shows an example of the results of Southern blotting. A) shows the results using 5'probe, B) 3'p
The result using robe is shown. When 5'probe was used, the wild-type mouse showed a 5.6 kb wild-type Doc2α gene band, the heterozygous mutant mouse showed two wild-type 5.6 kb and mutant 6.4 kb bands, The homozygous mutant mouse having the Doc2α mutant gene homozygously showed only the mutant 6.4 kb band. When 3'probe was used, wild-type mice showed a 8.6 kb wild-type Doc2α gene band, and heterozygous mice showed wild-type 8.6 kb and mutant-type Doc2α genes.
Two 6.6 kb bands were shown, and the homozygous mutant mice showed only the mutant 6.6 kb band.

Example 6 Expression and Purification of Doc2α Polypeptide (1) Expression and Purification of N-Terminal Polypeptide of Doc2α 90 amino acids (Do
In order to prepare a DNA fragment encoding the c2α N-terminal polypeptide, an expression vector pGEX-2T-Doc2α-N was constructed by the following method. Has BamHI and KpnI sites at both ends,
A Doc2α cDNA (BALB / c mouse cDNA library (manufactured by CLONTECH)) containing a 0.27 Kb DNA fragment containing 90 amino acids below the 9th Met from the translation start codon and having a translation stop codon downstream of it. (A vector incorporating the full-length 1.5 kb of Doc2α cDNA) as a template. The sequences of the two oligonucleotides used as PCR primers are shown in SEQ ID NOs: 4 and 5 in the sequence listing. The PCR reaction was performed at 94 ° C. for 5 minutes according to a conventional method, and then repeated 30 times at 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 2 minutes, and further at 72 ° C. for 5 minutes. . The reaction product was subjected to electrophoresis to collect a 270b band. Finally, 0.8 μg of DNA was obtained. After digestion of this amplified DNA fragment with BamHI, it was inserted into the BamHI site of pGEX-2T (Pharmacia) to obtain the expression vector pGEX-2T-Doc2α-N.

Using the Escherichia coli DH5α strain into which the expression vector pGEX-2T-Doc2α-N was introduced, 90 amino acids on the amino terminal side of Doc2α were expressed as a fusion protein with glutathione-S-transferase. The culture of Escherichia coli DH5α was performed in LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl / 1 L distilled water), and the induction of N-terminal polypeptide expression of Doc2α was induced by 0.1 mM IPTG (isopropyl-
β-D-thiogalactopyranoside) and culturing at 30 ° C. for 3 hours. E. coli after expression induction
PBS (-) (8 g NaCl, 0.2 g KCl, 2.9 g Na ₂ HPO ₄ , 0.2 g KH ₂ P
Were suspended in O ₄ / 1L distilled water) was subjected to ultrasonic treatment. Then, the mixture was centrifuged at 100,000 × g for 1 hour at 4 ° C., and the supernatant was separated with a glutathione Sepharose column (Pharmacia).
Company). After washing the column to which the Doc2α N-terminal polypeptide was bound with PBS, the Doc2α N-terminal polypeptide fusion protein was eluted and purified with an elution buffer (20 mM glutathione, 50 mM Tris-HCl (pH 8.0)).

(2) Expression and purification of recombinant Doc2α Expression vector pRset-Do encoding full-length mouse Doc2α
c2α was constructed in the same manner as in the above-mentioned vector (pGEX-2T-Doc2α) (1). However, using a Doc2α cDNA as a template, a 1.5 kb DNA fragment having BamHI and KpnI sites at both ends, including from the translation initiation codon to the translation termination codon,
Then, using an oligonucleotide having SEQ ID Nos. 6 and 7 in the sequence listing as primers, 0.2 μg of a 1.5 kb DNA amplified fragment was obtained, and this DNA fragment was digested with KpnI, and then pRset
(Invitorogen) inserted into the KpnI site and the expression vector p
Rset-Doc2α was obtained.

Using an Escherichia coli BL2l strain into which the expression vector pRset-Doc2α has been introduced, a fusion protein having oligohistidine at the amino terminus (FW Studier and BA Moffatt,
J. Mol. Biol., 189, 113-130 (1986)). Cultivation of the E. coli BL2l strain in LB medium was performed at 30 ° C.
For 3 hours. Then, E. coli was added to a phosphate buffer (20 mM
The suspension was suspended in sodium phosphate, 500 mM NaCl, pH 7.8) and subjected to sonication. Then, 100,000 × g, 1 hour, 4 ° C
And centrifuged under the same conditions.
(Invitrogen). The column to which the Doc2α protein was bound was placed in a phosphate buffer (20 mM sodium phosphate, 500
After washing with mM NaCl, pH 6.0, elution buffer (500 mM imidazole, 20 mM sodium phosphate, 500 mM NaCl, pH 6.
At 0), the Doc2α protein was eluted. Doc2 eluted
The α protein was dialyzed using 20 mM HEPES (pH 7.4) and 1 mM DTT, and then applied to a Mono Q column (0.5 × 5 cm) (manufactured by Pharmacia). A buffer (20 mM HEPES (pH 7.4), 1 mM DTT,
1% cholic acid) and B buffer (20 mM HEPES (pH 7.4),
Elution was performed with a gradient of NaCl salt concentration using 1 mM DTT, 1 M NaCl, 1% cholic acid), and the fraction containing the Doc2α protein was collected to obtain a final purified product.

Example 7 Preparation of Anti-Doc2α Polyclonal Antibody Purified Doc2α N-terminal polypeptide (90 amino acids) in PBS obtained in Example 6 (1) was conjugated with an equal amount of Freund's complete adjuvant (manufactured by DIFCO LABORATORIES). (Purchased from Wako Pure Chemical)
Mixed with rabbits (Japanese white seed,
Male) was immunized subcutaneously three times every three weeks, and one week after the final immunization, whole blood was collected from the carotid artery to obtain antiserum. For the first immunization, 1 mg of the fusion protein was used, for the second immunization, 500 μg, and for the third immunization, 10 μg.

The antibody titer was checked by Western blotting using the Doc2α protein having oligohistidine at the amino terminal obtained in Example 6 (2) according to a conventional method (see Maniatis, T. et al., Supra). Was performed.

The above antiserum was prepared according to a conventional method using glutathione.
After removing the anti-GST antibody by applying to -S-transferase (GST) -conjugated Sepharose 4B column, the antibody to Doc2α was purified by applying to the fusion protein-conjugated Sepharose 4B column used for immunization.

Example 8 Expression Analysis of Doc2α Protein Wild-type mouse, Doc2α gene heterozygous mouse, and
To examine Doc2α gene homozygous mutant mice for Doc2α protein expression, extract the crude synaptosome fraction,
The defect was confirmed by Western blotting.

(A) Preparation of Crude Synaptosome Fraction The cerebrum was isolated from two mice of each genotype (5 weeks old). Isolate the cerebrum with 10 volumes of extraction buffer (4 mM
Hepes-NaOH (pH7.4), 0.32M sucrose, 1mMEDTA, 5mM
Homogenized in EGTA, 0.1 mM PMSF, 10 μg / ml leupeptin) using a Teflon homogenizer. The homogenate was centrifuged at 800 × g for 10 minutes to separate a crude cell membrane fraction. Take the supernatant and add 9000 xg
For 15 minutes, and the crude synaptosome fraction was separated by precipitation. The precipitated crude synaptosome fraction was suspended in an appropriate amount of distilled water, and the protein
The total protein content was quantified using t (manufactured by BIO-RAD) to adjust to include 8 mg / ml protein. An equivalent amount of 8M urea was added to the adjusted crude synaptosome fraction, followed by incubation at 37 ° C. for 15 minutes to prepare a soluble crude synaptosome fraction.

(B) Western blotting The soluble crude synaptosome fraction containing a protein equivalent to 20 μg was subjected to 2 × SDS-sample buffer (0.25 M Tris-HCl (pH 6.
8), 10% SDS, 40% glycerol, 10% 2-mercaptoethanol). The SDS solution was electrophoresed on an SDS-polyacrylamide gel (10%), and the resulting gel was subjected to PVDF membrane (Immobilon, Millipor).
(e company). Anti-Doc obtained in Example 7 above
Western blotting was carried out using a 2α rabbit antibody and an anti-rabbit immunoglobulin-sheep polyclonal antibody conjugated to horseradish peroxidase (manufactured by Amersham Japan) according to a conventional method (see Maniatis, T. et al., Supra). The signal was detected using an ECL detection reagent (manufactured by Amersham Japan). FIG. 4 shows the obtained results.

FIG. 4 shows the expression of Doc2α protein in the crude synaptosome fraction of the brain tissue of wild-type, Doc2α gene heterozygous mutant mice, and Doc2α gene homozygous mutant mice. The crude synaptosome fraction of wild-type (+ / +) and Doc2α gene heterozygous mutant mice (+/-) contained 45
A kDa Doc2α protein was observed, but this protein was not detected in Doc2α gene homozygous mutant mice (− / −). From this, Doc2α gene homozygous mutant mice,
The Doc2α gene was knocked out by homologous recombination, and it was confirmed that the Doc2α protein of 45 kDa was not expressed.

Example 9 Electrophysiological Analysis of Doc2α Homo Mutant Mice To examine changes in neurotransmission in Doc2α homo mutant mice, brains were excised from mice of each genotype.
Hippocampal slices were prepared and analyzed using field potential measurement.

(A) Preparation of Hippocampal Slice Sections Doc2α homozygous mutant mice and wild-type mice (each
(5-7 weeks of age), the brain was removed, and the hippocampus was excised using tweezers and scissors. The excised hippocampus was cooled to about 4 ° C., and then artificial cerebrospinal fluid (ACSF: Aerated with a mixture of 95% oxygen and 5% carbon dioxide).
119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl ₂ , 1.3 mM MgSO ₄ , 1
mM NaH ₂ PO ₄ , 26 mM NaHCO ₃ , and 10 mM glucose) and cooled for about 3 minutes. The cooled hippocampus was fixed to the sample table using 7% agar, and the micro slicer (DTK-
1000, manufactured by Dosaka EM Co., Ltd.) to prepare slices having a thickness of 300 to 400 μm. The cut slice was transferred again to the ACSF using a calspipet, and kept at 35 ° C. for about 2 hours while being aerated with a mixed gas.

(B) Field potential measurement using hippocampal slices In a recording chamber, ACSF containing 75 μM Picrotoxin (AC
SF-Picro) was perfused, and the prepared hippocampal slice was placed there. A double bipolar electrode was formed with a 75 μm platinum iridium wire and used as a stimulation electrode. Pull a 2mm outer diameter cored glass tube (GD-2, manufactured by Narimosha) with a puller, and pull it inside with a tube.
A recording electrode was filled with 9% NaCl-2% pontamine sky blue liquid. Each electrode is held by a hydraulic three-dimensional manipulator for fine movement, and the stimulation electrode is placed on the hippocampus on the slice.
It pierced the Schaffer collateral fibers projected from field CA3. The recording electrode pierced the dendritic side of the pyramidal cell layer in the hippocampal CA1 area. 80μ for normal recording stimulus
Second constant current pulses were given at 10 second intervals. The induced post-synaptic response caused by this is recorded as a collective potential and amplified by an amplifier (Axopatch-1D, manufactured by Axon).
The amplified potential was processed by a computer and analyzed.

FIG. 5 shows the results of measurement of long-term potentiation (LTP), a typical plasticity in hippocampal CA1 area. ○ indicates the enhanced level of response of wild-type mice (12 cases), and ● indicates the Doc2α homozygous mutant mice (11
4 shows the level of enhancement of the response of Example). The length of the vertical line at each point is
Indicates the magnitude of the standard error. For wild-type mice, 100Hz
Induced post-synaptic response is 1.
LTP that was enhanced by 58 ± 0.06 fold was observed, but suppression of LTP strength was observed in Doc2α homozygous mutant mice (1.32
± 0.07 times). This indicates that the Doc2α protein plays a role in long-term potentiation of hippocampal CA1 field.

[0061]

According to the present invention, a mouse lacking the Doc2α gene is provided. Since this mouse lacks the Doc2α gene, it is useful as an experimental animal for studying the pathophysiology and causes of nervous system and endocrine system diseases involving Doc2α, and developing therapeutic methods.

[0062]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2255 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism name: mouse Sequence characteristics Characteristic symbol: CDS location : 361. . How to determine the 1575 features: S sequence TGGTGCAGAA GATGGCCAGA GCTTCAGAGC AAGCAGCAAA GGAGGGAAGC TGTGGGGTCC 60 CTGGAGGACT TGAGGGGATG GGGACAGAGG GAGAGCCCAG GATGAGAGGA AAACCCTGGA 120 AAGGTGACAG AGGGACAATG TGCAGGTAGT GAGCAGGGTT AAGGGGGAGT CACGACCTGT 180 CTGCTTTAGG ACTGATCTGG CAACCCACCC AGCCCTTTGT GAAGCCAGGC CTCCTGCCTG 240 CCCACCAGCC AGGGCAGATC ATCTTTTCCC TCGACACCCA GGAAGGGAGG GCAGTGAGGT 300 TCCTACAGAC CCCAGCCGGC CACTCCAGCT CTACACCCGT CTCCCAGTCA GAGGTGCTGC 360 ATG AGG GGC CGC AGG GGC GAT CGC ATG ACC ATC AAC ATC CAG GAG CAC 408 Met Arg Gly Arg Arg Gly Asp Arg Met Thr Ile Asn Ile Gln Glu His 5 10 15 ATG GCC ATC AAC GTG TGC CCT GGA CCC ATC AGG CCC ATC CGC CAG ATC 456 Met Ala Ile Asn Val Cys Pro Gly Pro Ile Arg Pro Ile Arg Gln Ile 20 25 30 TCC GAC TAC TTC CCT CGC AGG GGG CCA GGA CCA GAG GGT GGC GGC GGC 504 Ser Asp Tyr Phe Pro Arg Arg Gly Pro Gly Pro Glu Gly Gly Gly Gly 35 40 45 GGC GGT GGC ACG GGC TGC GGG GAA GCC CCA GCT CAT CTG GCC CCT CTG 552 Gly Gly Gly Thr Gly Cys Gly Glu Ala Pro Ala His Leu Ala Pro Leu 50 55 60 GCT CTG GCC CCC CCT GCG GCT CTC CTT GGG GCC ACT ACA CCC GAC GAT 600 Ala Leu Ala Pro Pro Ala Ala Leu Leu Gly Ala Thr Thr Pro Asp Asp 65 70 75 80 GGA GCT GAG GTA GAC AGC TAC GAC TCG GAT GAT ACC ACC GCC CTG GGC 648 Gly Ala Glu Val Asp Ser Tyr Asp Ser Asp Asp Thr Thr Ala Leu Gly 85 90 95 ACA CTG GAA TTT GAC CTT CTC TAT GAT CAG GCT TCC TGC ATG CTG CAC 696 Thr Leu Glu Phe Asp Leu Leu Tyr Asp Gln Ala Ser Cys Met Leu His 100 105 110 TGT AGA ATC CTC AGG GCC AAG GGC CTC AAG CCC ATG GAT TTC AAT GGC 744 Cys Arg Ile Leu Arg Ala Lys Gly Leu Lys Pro Met Asp Phe Asn Gly 115 120 125 CTG GCT GAC CCC TAT GTA AAG CTT CAC CTC CTG CCA GGA GCC TGC AAG 792 Leu Ala Asp Pro Tyr Val Lys Leu His Leu Leu Pro Gly Ala Cys Lys 130 135 140 GCC AAT AAG CTA AAA ACC AAG ACA CAG AGG AAC ACA CTG AAC CCT GTG 840 Ala Asn Lys Leu Lys Thr Lys Thr Gln Arg Asn Thr Leu Asn Pro Val 145 150 155 160 TGG AAT GAG GAG CTG ACG TAC AGC GGG ATC ACG GAT GAT GAC ATC ACC 888 Trp Asn Glu Glu Leu Thr Tyr Se r Gly Ile Thr Asp Asp Asp Ile Thr 165 170 175 CAC AAG GTG CTC AGG ATC TCT GTC TGT GAT GAG GAC AAG CTG AGC CAC 936 His Lys Val Leu Arg Ile Ser Val Cys Asp Glu Asp Lys Leu Ser His 180 185 190 AAT GAA TTC ATT GGG GAG ATC CGA GTG CCC CTC CGC CGC CTC AAG CCT 984 Asn Glu Phe Ile Gly Glu Ile Arg Val Pro Leu Arg Arg Leu Lys Pro 195 200 205 TCA CAG AAG AAG CAT TTT AAC ATC TGC CTT GAG CGC CAG GTC CCG CTT 1032 Ser Gln Lys Lys His Phe Asn Ile Cys Leu Glu Arg Gln Val Pro Leu 210 215 220 CCT TCA CCC TCT TCA ATG TCT GCG GCG CTG AGG GGC ATA TCC TGT TAC 1080 Pro Ser Pro Ser Ser Met Ser Ala Ala Leu Arg Gly Ile Ser Cys Tyr 225 230 235 240 CTG AAG GAG CTG GAG CAG GCA GAG CAG GGA CCT GGG CTG CTG GAA GAG 1128 Leu Lys Glu Leu Glu Gln Ala Glu Gln Gly Pro Gly Leu Leu Glu Glu 245 250 255 CGC GGC CCG ATC CTG CTG AGC CTC AGC TAC AGC TCT CGG CGG CAT GGG 1176 Arg Gly Pro Ile Leu Leu Ser Leu Ser Tyr Ser Ser Arg Arg His Gly 260 265 270 CTG CTG GTG GGC ATT GTT CGC TGT GCG CAC CTG GCT GCA ATG GAT GTT 1224 Leu Leu Val G ly Ile Val Arg Cys Ala His Leu Ala Ala Met Asp Val 275 280 285 AAC GGC TAC TCT GAC CCT TAT GTG AAG ACG TAC TTG AGA CCA GAT GTG 1272 Asn Gly Tyr Ser Asp Pro Tyr Val Lys Thr Tyr Leu Arg Pro Asp Val 290 295 300 GAT AAG AAA TCC AAG CAC AAA ACA TGT GTA AAG AAG AAG ACA CTA AAT 1320 Asp Lys Lys Ser Lys His Lys Thr Cys Val Lys Lys Lys Thr Leu Asn 305 310 315 320 CCG GAA TTT AAT GAG GAA TTC TTC TAT GAG ATT GAA CTC TCC ACT CTG 1368 Pro Glu Phe Asn Glu Glu Phe Phe Tyr Glu Ile Glu Leu Ser Thr Leu 325 330 335 GCC ACT AAG ACC CTG GAG GTC ACA GTC TGG GAC TAC GAC ATT GGC AAA 1416 Ala Thr Lys Thr Leu Glu Val Thr Val Trp Asp Tyr Asp Ile Gly Lys 340 345 350 TCC AAT GAC TTC ATA GGT GGT GTG TCT CTG GGG CCA GGA GCC CGG GGA 1464 Ser Asn Asp Phe Ile Gly Gly Val Ser Leu Gly Pro Gly Ala Arg Gly 355 360 365 GAG GCC CAG AAA CAC TGG AAT GAC TGT CTA CAT CAG CCG GAC ACA GCC 1512 Glu Ala Gln Lys His Trp Asn Asp Cys Leu His Gln Pro Asp Thr Ala 370 375 380 CTG GAG CGC TGG CAT ACT CTG ACC AGC GAG CTG CCC CCT GCA GCA GG G 1560 Leu Glu Arg Trp His Thr Leu Thr Ser Glu Leu Pro Pro Ala Ala Gly 385 390 395 400 GCT TAC CCT TTG GCT TGA GTGAACAGCA GCTGTCCATC ACAGGCCCTC TGGGGCCGGG 1619 Ala Tyr Pro Leu Ala 405 TCCAGTACCC AACCTTCGCA CGAGTGTGTT GCACGTTTAC ATGGGGTGGG ATGCCCCTGC 1678 CCCACACTAC CTGTCTTATT TTTGTGAGTC TCTGTGACGG GCCTGTCTTC TTGTGAGGGA 1738 CTGTGGAGAG CTATATTCAC ATATGCAAAC TTCCTCCTGC CTGCCTTGCT GTTACCTGCA 1798 AATATGCAAC CACCCGTGCA CAGGGCCACG TGGGAGTCTC CTGCCAGTGC CCCACCCCAT 1858 CCTGCAGCTC CTTTCTTGGC CTTTCCAGGC TGCCTGGTCC CCTGCCCCAC AGGGAGGGGG 1918 TCGGATTAGG GAAGATTAGA GGAGGACGTT TCCAAATAGA GGAAAGGACA AGGAAAGAAA 1978 GAGCCAACTC TTTAATACAG AGCCTTCACT AAATCCCAGC CCTGCGTAGC TGCTCTTCTA 2038 AAGGGGCGGC CACCGTAGGT CTCTACCTCC CGAAGATGTA GCAGACCCTT TTGGCCACTC 2098 GTTTAAATAA CTGTTCCCTG GATGGGGCTG GGATGTAAGG GCAGGACCCT GCCACCTTCC 2158 TCGGGGACAT TGTGCATGTT TACGCCTTTT CTGATTGTGT CAGTGGCCGA AGCATGGCAA 2218 CTGGGCATCA ATAAACATCT CTTGAGGAAA AAAAAAA 2255

SEQ ID NO: 2 Sequence length: 33 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: CAATGGCCTG GACTGACCCC TATGTAAAGC TTC 33

SEQ ID NO: 3 Sequence length: 33 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA Sequence: ACTCGGATCT CCCCAATGAATTTCATTGTGGCTC
33

SEQ ID NO: 4 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: CATGGATCCG GTACCATGAC CATCAACATC CAGGAG 36

SEQ ID NO: 5 Sequence length: 32 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: TTGGGATTCT AGGCGGTGGC ATCATCCGAG TC 32

SEQ ID NO: 6 Sequence length: 36 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: CATGGATCCG GTACCATGAG GGGCCGCAGG GCGATC 36

SEQ ID NO: 7 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence: CATGGATCCG GTACCTCAGG CTGAGGACAG AGCCCC 36

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing mouse and human Doc2α proteins. FIG. 1A shows mouse Doc2α protein, and FIG. 1B shows human Do
1 shows the c2α protein. Mid on the N-terminal side is a domain that interacts with the Munc13-1 protein. C2 of the hatched box
Indicates the C2 region. The numbers indicate the positions of the amino acid residues.

FIG. 2: Mouse Doc2α gene, vector for homologous recombination,
FIG. 2 is a schematic diagram showing homologous recombinants of Doc2α gene and Doc2α gene. Where B indicates Bam HI; G indicates Bgl II; E
Indicates Eco RI; H indicates Hind III; and S indicates Sph
Indicates I. However, all sites are not shown for the Sph I site. A) shows a restriction map of mouse Doc2α gene. Black boxes indicate exons, and solid lines between exons indicate introns. ATG in the box indicates the start position of translation, and STOP indicates the start position of translation. Bold lines indicate 5 'and 3' probes for Southern blot analysis.
The restriction enzyme sites used for construction of the Doc2α gene are shown in the figure. B) shows a vector for homologous recombination, which is an example of an insertion construct targeting the Doc2α gene of a mouse. Black boxes indicate exons of the Doc2α gene, and solid lines between exons indicate introns. β-lactamase gene (l
ac-Z), neomycin resistance gene (pgk-Neo), and diphtheria toxin A fragment (DT-A) gene are indicated by arrows. The direction of the arrow indicates the direction in which the gene is transcribed. Solid lines indicate homologous regions. C) Schematic diagram of the structure of the gene of the homologous recombinant in which the Doc2α gene has been modified.

FIG. 3 is an electrophoretic photograph showing the result of Southern blot analysis of mouse DNA. FIG. 3A shows the result using 5 ′ probe, and FIG. 3B shows the result using 3 ′ probe. The position of the wild-type Doc2α gene band when using 5'probe (5.6k
b) and the position of the 6.4 kb band of the Doc2α-deficient gene into which the neomycin gene was inserted. Position of wild-type Doc2α gene band when using 3'probe (8.6 kb)
And the position of the 6.6 kb band of the Doc2α-deficient gene into which the neomycin gene was inserted.

FIG. 4 is an electrophoretic photograph showing the results of Western blot analysis of the expression of Doc2α protein in crude synaptosome fractions of brain tissues of wild-type, Doc2α gene heterozygous mutant mice, and Doc2α gene homozygous mutant mice.

FIG. 5 is a graph showing the results of comparing long-term amplification in the hippocampal CA1 field between wild-type and Doc2α homozygous mutant mice.

Claims (4)

[Claims] 1. A transgenic mouse lacking the Doc2α gene. 2. The DoC is obtained by replacing or inserting any partial sequence of the Doc2α gene with another gene.
The mouse according to claim 1, wherein the c2α gene is deleted. 3. The mouse according to claim 2, wherein the other gene is a selectable marker gene. 4. The mouse according to claim 1, wherein a selectable marker gene is substituted or inserted into a partial sequence including a part of the first exon of the Doc2α gene.