JPH1132627A - Mammalian animal of which sez-6 gene expression is suppressed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject mammalian animal capable of screening a new anticonvulsive agent and useful as a disease model for developing a new treating method by inactivating SEZ 6 gene. SOLUTION: This objective mammalian animal is obtained by inactivating SEZ-6 gene. Inactivation of the SEZ-6 gene is performed by constructing a targeting vector, incorporating the obtained targeting vector into undifferentiated cells having a complete embryological performance, isolating the cells in which the SEZ-6 gene is inactivated by a homologous recombination, injecting the isolated cells into an animal embryo, obtaining a chimeric animal which can be mated with a normal animal, by transplanting the embryo into a uterus of a temporary mother animal. Also, the mammalian animal can be selected from a mouse, a rat and a rabbit, and further, the mammalian animal can be made as an animal with convulsive disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel mammal in which the expression of the SEZ-6 gene is suppressed, and a method for screening an anticonvulsant using the mammal.

[0002]

2. Description of the Related Art Epilepsy responds to cerebral dysfunction caused by seizures. Clinically, it depends on the location and extent of seizures in the brain. Defined. As for anticonvulsants used for the treatment of epilepsy, many drugs (barbitur, hydantoin, acetylurea, etc.) have been developed, but long-term administration causes side effects such as gingival hyperplasia, rash and visual impairment. It is known from electrophysiological analysis that seizures associated with epilepsy are caused by transient abnormal and excessive excitation of the nervous system.However, at the level of gene expression, It is not clear if the excitement occurs in nerve cells.

[0003] However, studies on convulsion generators have been conducted mainly using electrophysiological techniques, and no attempt has been made to clarify the mechanism of convulsions at the molecular level. Furthermore, there has never been an approach for cloning a gene whose expression level changes by administration of a convulsant-inducing agent and elucidating the mechanism of convulsions at a molecular level. The present invention makes the above-mentioned approach, and the technical problem itself is already novel.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have a technical problem of elucidating the mechanism of convulsions by a molecular biological technique by genetic manipulation, not by a conventional electrophysiological technique. More specifically, the specific technical issues of elucidating the mechanism of convulsions at the molecular and individual levels, developing new therapeutic drug development systems, and developing model animals to evaluate them are newly added. Set to.

The inventors of the present invention have studied from various directions in order to solve the technical problems newly set as described above, and as a result, have focused on the gene SEZ-6 for the first time.

[0006] In a culture system, the gene SEZ-6 is used as a convulsant-inducing agent, pentylenetetrazole (p
(Shimizu-Nishika) is a convulsions open gene cloned as one of the genes whose expression level is increased by (entrynetrazole).
wa, K .; et al. Mol. Brain Re
s. , 28, 201-210, 1995). As a result of molecular biological analysis, it was found that the SEZ-6 gene encodes a membrane protein that is specifically expressed in the brain. In addition, the expression of the SEZ-6 gene is deeply involved in the occurrence of seizures, because it is found in the brain of adult mice, such as the hippocampus, cerebral cortex, and basal ganglia, which are relatively easy to focus on epilepsy. For the first time. Therefore, the present inventors have obtained a novel idea that it is necessary to divide a mammal in which the expression of the SEZ-6 gene is suppressed in order to solve the above technical problem.

Furthermore, the present inventors have found that producing a mammal in which the expression of the SEZ-6 gene is suppressed not only leads to elucidation of the mechanism of convulsion at the molecular and individual levels, but also The present inventors have further obtained a new idea of providing a method of screening for a new anticonvulsant using the mammal, and a disease model animal useful for developing a new therapeutic method.

[0008] Accordingly, the present invention provides SEZ- useful for elucidating the mechanism of convulsions at the molecular or individual level.
It is an object of the present invention to provide a mammal in which the expression of six genes is suppressed.

Another object of the present invention is to provide a mammal in which the expression of the SEZ-6 gene is suppressed, which is useful for a method for screening anticonvulsants and for developing a new therapeutic method.

[0010]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve the above object, and as a result, have found that SEZ-6.
Based on the basic technical concept of a mammal in which the expression of the gene is suppressed by inactivating the gene, the present invention has been completed on a model mammal for convulsion suppression and a method for screening an anticonvulsant using the same. Hereinafter, the present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION As mammals used in the present invention, for example, mice, rats, rabbits and the like can be mentioned, but other mammals can also be widely used.

The mammal of the present invention in which the expression of the SEZ-6 gene is suppressed is produced, for example, through the following steps.

(1) Identify and isolate the SEZ-6 genomic gene of the mammal. (2) In order to inactivate the SEZ-6 gene of the mammal, a targeting vector is constructed by inserting a marker gene into the identified gene. (3) This targeting vector is introduced into an undifferentiated cell having developmental totipotency, such as an ES cell. (4) Isolate ES cells in which the SEZ-6 gene has been inactivated by homologous recombination. (5) The isolated ES cells are injected into an animal embryo, and transplanted into the uterus of a foster mother to obtain a chimeric animal. The chimeric animal is bred to a normal animal to obtain a heterozygous animal (+/-), and the heterozygous animal is bred to each other to obtain a homozygous animal (-/-), ie, the SEZ-6 allele. Are inactivated to obtain a mammal in which the expression of the SEZ-6 gene is suppressed.

Among the above steps, the first step, identification and isolation of the SEZ-6 genomic gene, is performed by S
EZ-6 gene cDNA (Shimizu-Nishi
kawa, K .; et al. Mol. Brain Re
s. , 28, 201-210, 1995) as a probe and screening a genomic DNA library derived from mammals.

The construction of the targeting vector, which is the second step, can be performed as follows. The purpose of the targeting vector is to introduce a marker gene, for example, a neomycin resistance gene (neor) into the transcription regulatory region or at least one exon portion of the target gene, SEZ-6 gene, by homologous recombination.
It consists in inactivating the EZ-6 gene. Here, homologous recombination means recombination that occurs based on the homology of the base sequence between the targeting vector and the corresponding gene on the chromosome. In order to cause this homologous recombination, it is necessary that the marker gene has a region (homologous region) having the same base sequence as a part of the target gene to be inactivated. Occurs,
The marker gene is inserted at the target site of the target gene,
While destroying the function of the target gene, selection (positive selection) for selecting only ES cells into which the marker gene has been introduced becomes possible. Furthermore, in reality, since non-homologous recombination occurs more frequently than homologous recombination, another marker gene, such as diphtheria toxin A fragment (DT-A), is located outside the homologous region in the targeting vector. Connect. By utilizing the fact that ES cells into which the DT-A gene has been introduced are lethal, non-homologous recombination ES cells can be removed. In this way,
By performing positive-negative selection, a homologous recombinant can be efficiently obtained.

[0016] The third step, the introduction of the targeting vector into ES cells, can be performed according to a method using electroporation or the like. ES cells into which the targeting vector is introduced need to be cultured and maintained in an undifferentiated state so as not to lose the ability to differentiate into germ cells. To this end, appropriate vegetative cells are added to the medium as feeder cells, and human LIF (Leukemia Inhibitor, a differentiation inhibitory factor) is further added.
y Factor). In addition, fetal calf serum, nucleosides, non-essential amino acids, 2-mercaptoethanol, etc. are appropriately added to the medium. As a vegetative cell, S
TO cells or mouse fetal fibroblasts are suitable. The addition density of the vegetative cells is about 1 × 10 ⁶ cells / 35 mm d
ish. Addition concentration of human LIF is a 10 ³ units / ml may be higher. The addition concentration of fetal bovine serum is 20% and the addition concentration of nucleoside is 10 to 30 n.
M. The non-essential amino acid is preferably a non-essential amino acid solution manufactured by Gibco, and its concentration is 0.05
mM. The concentration of 2-mercaptoethanol is 0.1
mM. In order to introduce a targeting vector into ES cells by the electroporation method, the ES cells are suspended in a buffer, treated with an appropriate restriction enzyme, and a linearized targeting vector is added. Electroporation is performed using a suitable electroporation apparatus under the conditions of 250 V / 0.4 cm, 480 mF. The restriction enzyme is selected in consideration of the restriction enzyme site of the targeting vector.
After the introduction of the targeting vector, the ES cells were cultured in the above medium for about 24 hours.
Replace with medium containing 0 mg / ml G418. G41
It is preferable that the 8-supplemented medium be replaced with a new one every day, and the culture is continued for about one week.

In the fourth step, drug-resistant ES cells obtained by introducing the targeting vector are isolated. Whether or not the isolated ES cells have actually undergone homologous recombination can be confirmed using PCR and Southern hybridization. That is, first, PCR
As a primer for the method, a primer is synthesized from the nucleotide sequence contained in the targeting vector and the nucleotide sequence present on the SEZ-6 gene not contained in the targeting vector. Then, using these primers, it is confirmed that the DNA fragment in the region is amplified. PC
From the ES cells in which homologous recombination was confirmed to have occurred by the R method, restriction enzyme fragments capable of distinguishing mutant alleles from wild-type alleles were further confirmed by Southern hybridization. The extracted DNA is treated with a restriction enzyme and subjected to agarose electrophoresis, followed by Southern hybridization using SEZ-6 gene DNA as a probe. When homologous recombination by the targeting vector occurs, the site of cleavage by the restriction enzyme changes, so that in the cell in which homologous recombination has occurred, a DNA fragment having a size different from that of a wild-type cell is detected. It can be confirmed whether or not the problem has occurred.

The fifth step, the production of a germ-line chimeric animal and a mammal in which the expression of the SEZ-6 gene is suppressed, is performed as follows. First, homologous recombination ES cells are injected into an animal embryo, cultured in vitro, and then transplanted into the uterus of a foster parent. Here, in the case of a mouse, an embryo in a developmental stage from an 8-cell stage embryo to a blastocyst is used because an animal embryo is suitable for chimera formation. ES
Usually, 10 to 5 cells are injected. The embryo into which the ES cells have been injected is cultured in vitro and then transplanted to a foster animal. A chimeric animal having a high ES cell contribution rate is likely to be a germline chimeric animal, but by crossing the chimeric animal with a normal animal, it can be confirmed that the chimeric animal is a germline chimeric animal. In this way, a heterozygous animal that is a germline chimeric animal is obtained,
By mating heterozygotes, homozygous animals in which both SEZ-6 alleles have been inactivated by the targeting vector can be obtained.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0020]

【Example】

Example 1: Preparation of mouse in which expression of SEZ-6 gene was suppressed (1) Isolation of mouse SEZ-6 genomic gene Mouse SEZ-6 gene cDNA (Shimizu-N
Ishikawa, K .; et al. Mol. Brai
n Res. , 28, 201-210, 1995) as a probe and screening from a 129SVJ-derived genomic gene library (manufactured by STRATAGENE).

(2) Construction of Targeting Vector The construction of the targeting vector SE will be described with reference to FIG. As the homologous region of the 5 'side Kpn I- Sca I
About 7.6 kb of the fragment was used. Downstream of the 5 'homology region is a PGK-1 promoter (obtained from Dr. Shinichi Aizawa, RIKEN) and a neomycin resistance gene (neo).
r) (manufactured by STRATAGENE) was used as a positive selection marker. The 3 'homologous region is
The 1.3 kb Sca I- Kpn I fragment was
r downstream. The MC-1 promoter (manufactured by STRATAGENE) and the DT-A gene (obtained from Dr. Shinichi Aizawa, RIKEN) were connected as a negative selection marker downstream of the 3'-side homologous region.

[0022] The homologous recombination result, neo r is inserted into the Sca I site in the SEZ-6 gene exons (103-763). The mRNA transcribed from this mutant SEZ-6 gene is predicted to be translated into a short protein of 172 amino acids due to the presence of a stop codon in front of the PGK promoter (a 977 amino acid protein from the wild-type SEZ-6 gene). Will be translated). This short protein lacks the functional domain structure of the SEZ-6 gene product revealed by homology search, and is considered to have no function in vivo.

(3) Electroporation of targeting vector to CCE cells CCE cells are ES cells established from blastocysts of 129Sv // Ev mice (Kuehn, MR.e).
t al. , Nature, 326, 295-298,
1987). CCE cells were obtained by using mouse fetal fibroblasts transformed with neor as a feeder (1 × 1).
0 ⁶ cells / 35 mm culture dish), added the human LIF (10 ³
Unit / ml), 20% fetal calf serum, nucleoside,
Dulbecco's modified medium containing a non-essential amino acid, 2-mercaptoethanol (Hirofumi Suemori, Norio Nakatsuji, Experimental Medicine, vo
l. 7, 293-298, 1989). CC
E cells were suspended in 800 ml of PBS (pH 7.0), a targeting vector linearized with AatII was added (50 μg), and electroporated at 250 V / 0.4 cm, 480 mF using a Bio Rad Gene Pulser. After electroporation, the cells were cultured under the above conditions for 24 hours, and thereafter, 200 mg / ml G4
The medium containing 18 was replaced. Fresh G every day
The medium was replaced with a 418-added medium, and the culture was continued for 7 to 9 days.

(4) Identification of homologous recombinants G418-resistant clones were picked up and grown. A part was stored frozen in liquid nitrogen, and DNA was extracted from the rest. Identification of the homologous recombinant was performed by PCR and Southern hybridization. PCR was performed using 1 pmol / ml NEO16 primer 5'-TCC --- ATT-3 '(SEQ ID NO:
1), 1 pmol / ml as antisense primer
SE4 primer 5'-GCT --- CCC-3 '
(SEQ ID NO: 2) under the following reaction solution and temperature conditions, using PERKIN ELMER Gene Ampsy.
Performed using a stem 9600.

[Reaction liquid composition] 10 mM Tris-HCl (pH 8.3) 50 mM KCl 4 mM MgCl ₂ 0.2 mM dATP, dGTP, dCTP, dTTP 0.1 U / ml Taq polymerase (manufactured by TAKARA) [Temperature conditions] 94 ° C for 30 seconds, 70 ° C for 2 minutes 30 seconds (30 cycles)

The length of the DNA fragment amplified under these conditions was about 1.5 kb. Next, the DNA of the ES cells whose amplification was confirmed by PCR was replaced with ScaI or PstI.
And agarose gel electrophoresis was performed, and Southern hybridization was carried out using a Kpn I- Pst I fragment (about 1.7 kb) downstream of the 3 ′ homologous region as a probe . In the wild-type allele, 4.7kb in Sca I digestion,
A 3.1 kb fragment was observed in the Pst I digestion, but in the clone of the homologous recombinant, Sca I digestion was 4.7 kb in addition to 4.7 kb, and Pst I digestion was 3.1 kb and 3.8 kb in addition to 3.1 kb. Was also observed. This corresponds to the Sc of the SEZ-6 gene as shown in FIG.
This is because neo is inserted into a I site. This proved that this clone underwent homologous recombination with the targeting vector at one allele.

(5) Preparation of chimeric mouse and SEZ-6 gene-deficient mouse By targeting as described above, one of the SEZ-6
The CCE cells in which the gene was inactivated were injected into C57BL / 6 mouse blastocysts and transplanted into the uterus of a foster mother. Where C
10 to 15 CE cells were injected. As a result, 60 embryos were transferred to 4 foster parents, and 11 offspring were obtained. Eight of them were chimeric mice based on coat color (five males and three females). When the chimeric mouse and the C57BL / 6 mouse were bred, four were confirmed to be germline chimeras, and a heterozygous mouse in which one of the SEZ-6 alleles was inactivated by targeting was obtained. Male and female heterozygous mice were crossed to obtain homozygous mice.

[0028]

The homozygous mammal in which both SEZ-6 alleles obtained by the present invention are inactivated is useful as a model animal for convulsive disease. In addition, efficient screening of anticonvulsants and development of new therapeutic agents are possible using the mammal or somatic cells derived from the animal.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCGTGCTTTA CGGTATCGCC GCTCCCGATT 30

SEQ ID NO: 2 Sequence length: 30 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GCTATTGCTG GTAGTGCACA GGCAGAACCC 30

[Brief description of the drawings]

FIG. 1 shows a restriction map of a mouse SEZ-6 genomic gene and a targeting vector (SE).

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Keiko Nishikawa, Inventor 26-9 AST EP Saijo 202, Saijo Nishihonmachi, Higashihiroshima City, Hiroshima Prefecture (72) Inventor Eiichi Sugaya 4-10-8, Kichijoji Minamimachi, Musashino City, Tokyo

Claims (6)

[Claims] 1. A mammal in which the expression of a SEZ-6 gene is suppressed by inactivating the gene. 2. In order to inactivate the SEZ-6 gene,
Constructing a targeting vector; introducing the resulting targeting vector into a developmentally totipotent, undifferentiated cell; isolating a cell in which the SEZ-6 gene has been inactivated by homologous recombination; The mammal according to claim 1, wherein the obtained cells are injected into an animal embryo and transplanted into the uterus of a foster parent to obtain a chimeric animal; and the obtained chimeric animal is bred with a normal animal. . 3. The mammal according to claim 1, wherein the mammal is selected from a mouse, a rat, and a rabbit. 4. The mammal according to claim 1, wherein the mammal is a model mammal for a convulsive disorder.
The mammal according to Item. 5. A method for screening an anticonvulsant, comprising using the mammal according to any one of claims 1 to 4. 6. A mammal in which expression of the SEZ-6 gene is suppressed by inactivating the SEZ-6 gene,
The anticonvulsant drug according to claim 5, comprising (1) administration of a convulsant-inducing agent, (2) administration of an anticonvulsant drug candidate, and (3) measurement of the anticonvulsant action of the candidate substance. Screening method.