KR19990075173A - Epilepsy-induced Mutant Mice and Methods for Preparing the Same - Google Patents

Abstract

The present invention relates to a mutant mouse induced epilepsy, and more particularly, to a mutant mouse in which the phospholipase Cβ1 (PLCβ1) gene is deleted and a method of producing a mutant mouse using gene targeting.

Description

Epilepsy-induced Mutant Mice and Methods for Preparing the Same

The present invention relates to a mutant mouse induced epilepsy, and more particularly, to a method of manufacturing a mutant mouse which lacks the phospholipase C-β1 gene and a mutant mouse using gene targeting.

Epilepsy is a neurological condition in which chronic and recurrent seizure occurs repeatedly or abnormally due to abnormal depolarization of brain cells, and is classified into local epilepsy and systemic epilepsy. In particular, systemic epilepsy is characterized by tonic-clonic or stiffening throughout the body, obstructing normal life and severely causing epilepsy convulsions frequently and repeatedly without consciousness recovery between seizures. The status epilepticus continues, leading to death in severe cases. Nevertheless, the treatment of systemic epilepsy is very difficult, because most of the mechanisms of epilepsy are not elucidated and the therapeutic agents that can be used are limited. Therefore, it is very urgent to understand the mechanism of epilepsy and to develop epilepsy treatments accordingly.

Epilepsy-induced experimental animal models are very useful for understanding epilepsy mechanisms and developing epilepsy treatments. The experimental animal models used to date have been induced by epistimulation or by the administration of drugs such as metrazole and kainic acid. These models observed epilepsy induced in an artificial environment. Only brain tissue changes were observed, not because of genetic traits, but there is a limit in understanding the mechanism of epilepsy, and it is difficult to develop epilepsy treatments based on this. There are disadvantages such as accompanying defects.

Accordingly, new models involving genetic epilepsy are needed to understand the epilepsy mechanisms of humans and to develop therapeutics for epilepsy.

Gene targeting studies the inherent function of a gene that has been destroyed at the individual level through the introduction of a target vector into a particular gene on a chromosome to destroy that particular gene in the genome and then through the pathology of the organism with that destroyed gene. In particular, when a destroyed gene is associated with epilepsy, the animal that is hit by this gene can be very useful as an experimental animal model for understanding epilepsy mechanisms and developing epilepsy therapeutics.

On the other hand, PLCβ protein is an important protein that is connected to the receptor, G protein, etc. and serves to transmit signals outside the cell into the cell, there are four isoenzymes of β1 to β4. PLCβ1 consists of 1216 amino acids and has a PH domain similar to plecstrin and an X domain and a Y domain, which are catalytic domains, as shown in FIG. 1A. The structure of the gene for this PLCβ1 protein is shown in Fig. 1B. However, the in vivo role of PLCβ1 protein is not known so far, and further, the relationship between PLCβ1 protein and epilepsy has not been reported yet.

The present inventors have found that mutant mice lacking the PLCβ1 gene show epilepsy to complete the present invention.

Accordingly, it is an object of the present invention to provide a mutant mouse in which epilepsy is induced.

Another object of the present invention to provide a method for producing the mouse.

FIG. 1A shows the structure of the PLCβ1 protein, FIG. 1B shows the structure of the PLCβ1 gene, FIG. 1C shows the hit vector of the PLCβ1 gene, and FIG. 1D shows the structure of the variant PLCβ1 gene hit with the hit vector.

Figure 2 is the result of Southern blot for the variant mice according to the present invention.

Figure 3 is a result of the polymerase chain reaction (PCR) for the variant mice according to the present invention.

4 is a result of Northern blot for the brain of the variant mice according to the present invention.

5 is a result of Western blot for the cerebral and cerebellum of the variant mice according to the present invention.

Figure 6 is a graph showing the survival rate over time after birth of the variant mice according to the present invention.

Figure 7 is a picture showing the epilepsy of the variant mice according to the present invention.

8a to 8d are graphs showing the high susceptibility of mutant mice according to the present invention for chiinic acid or metrazol.

9a to 9f are photographs showing the results of immunohistochemical analysis for the hippocampus of the variant mice according to the present invention.

10 is a graph showing phosphoinositide hydrolysis induced by CCh, ACPD or mCPP.

In accordance with the above object, in the present invention, a homozygous variant mutant PLCβ1 gene having a deletion at one site selected from the base sequence of the PLCβ1 gene corresponding to the amino acid sequence from the N-terminal to the PH domain of PLCβ1 , Mus muculus mutant mice induced with epilepsy are provided.

In addition, in the present invention (1) introducing a hit vector for a site selected from the nucleotide sequence of the PLCβ1 gene corresponding to the amino acid sequence from the N- terminal to the PH domain of PLCβ1 into the embryonic stem cells, (2) the embryonic stem cells Obtaining chimeric mice by injecting into the blastocyst of the blastocyst, (3) crossing the chimeric mice to obtain heterozygote mice, and (4) crossing the heterozygous mice to obtain homozygous variant mice. There is provided a method of producing the variant mouse comprising the step of obtaining.

Hereinafter, the variant mice of the present invention will be described in detail.

In the present invention, "a variant PLCβ1 gene in which a deletion occurs at a site selected from the base sequence of the PLCβ1 gene corresponding to the amino acid sequence from the N-terminal to the PH domain of PLCβ1" is a base essential for phospholipase function by destroying the site. Refers to a null variant PLCβ1 gene (hereinafter referred to as the “variant PLCβ1 gene”) in which the region from the PH region to the 3 ′ end, which contains sequences, is not expressed at all, ie is deleted at that site. .

The mutant mice of the present invention are homozygotes having the PLCβ1 ^-/- genotype of defects in the same pair of homologous chromosomes, and are normally born, but due to the stiffness-moderation or stiffness expansion of the entire body. There is a characteristic with a phenotype showing systemic epilepsy characterized.

Mutant mice of the present invention start to die from about 20 days after birth due to epilepsy, and the survival rate rapidly decreases with time, and for this reason, reproduction is impossible. In contrast, heterozygous mutant mice having a genotype of PLCβ1 ^+/- (hereinafter referred to as heterozygous mice) are similar to those of wild type mice having a genotype of PLCβ1 ^{+ / +} (hereinafter referred to as 'normal mice'). Equally growing normally, with reproductive phenotypes, no epilepsy is expressed.

In addition, the mutant mice of the present invention have very high sensitivity to epileptic inducers such as metrazole and chinic acid, so that epilepsy is induced by a small amount of epilepsy not induced in normal mice.

As a result of immunohistochemical analysis that the interneuron containing somatostatin is selectively lost in the hilus of the hippocampus of the brain in the mutant mouse of the present invention, the PLCβ1 protein is a muscarinic acetylcholine ( Experimental results of the present inventors showing an important role in signaling by muscarinic acetylcholine receptors and reports that electrical stimulation of adjacent granule cells in the hippocampus results in the loss of intermediate neurons (Sloviter, RS, Science, 235, 73-76). The epilepsy mechanism of the mouse of the present invention inferred from (1987)) is that the hippocampus is overexcited by the lack of PLCβ1 protein during the signaling process by muscarinic acetylcholine in the hippocampus of the brain. become hypersensitive and eventually lose the intermediate neurons containing somatostatin at the gate of the hippocampus, causing epilepsy It seems to be.

Hereinafter, the method for producing a variant mouse of the present invention will be described in detail.

First, a hit vector is prepared for a site selected from the nucleotide sequence of the PLCβ1 gene corresponding to the amino acid sequence from the N-terminus to the PH domain of PLCβ1. At this time, the hit vector includes two homologous fragments, a PGK-neo cassette, an SV40 poly (A) sequence, and a thymidine kinase (thymidine) located at a portion of the nucleotide sequence from the PLCβ1 gene initiation site to the PH domain. A vector containing a kinase gene cassette can be used, wherein homologous recombination takes place in the homologous fragment, the PGK-neo cassette is inserted into a normal exon, and the SV40 poly (A) sequence destroys the PLCβ1 gene. Causing mutations, thymidine kinases play a role in enhancing gene targeting efficiency. Representative examples of the hit vector include the hit vector pDS-5 of FIG. 1C (the PGK-neo cassette and the SV40 poly (A) sequence are inserted at the PLCC1 gene exon region and the thymidine kinase gene cassette is located at the 3 'end). Can be. As a method for constructing a hit vector, a conventional method may be used. For example, the hit vector pDS-5 is attached with a thymidine kinase gene cassette at the 3 'end of a genomic DNA fragment containing an exon of the PLCβ1 gene, and a PGK in the middle of the exon. -Neo cassette is produced by inserting.

The targeting vector is introduced into mouse embryonic stem cells. At this time, as embryonic stem cells, embryonic stem cell line J1 (prepared from MIT's R. Jeananisch) was transferred to ES medium [15% fetal bovine serum (Hyclone Co.), 1x penicillin-streptomycin (Gibco Co.), 1x non-essential. Amino acid (Gibco Co.), 0.1 mM 2-mercaptoethanol, DMEM medium (Gibco Co.)] and incubated for 2 to 3 days at 37 ℃ and then the germ cell group in 1 mM EDTA solution containing 0.25% trypsin To separate into single cells. The isolated single embryonic stem cells and the hit vector are subjected to electroporation under conditions at 250V / 500 kV to 270V / 500 kV, preferably 270V / 500 kV. Embryonic stem cells into which a targeting vector has been introduced can be selected by incubating for 5 to 7 days in an ES medium containing an appropriate antibiotic, and in the case of pDS-5 in a medium containing 0.3 mg / ml G418 and 2 μM of liver cyclovir. Can be screened. Selected targeted embryonic stem cells can be identified by a conventional method such as Southern blot.

The structure on the chromosome hit with the hit vector pDS-5 is shown in FIG. 1D. In FIGS. 1B, 1C and 1D, B represents BamHI, Bg represents BglII, E represents EcoRI, X represents XbaI, XI represents XhoI, and S represents SacI, the black box shows the exon hit and the white box shown below the normal gene The probe used for Southern blot is shown.

Embryonic stem cell clones identified with the PLCβ1 gene hit were treated with trypsin at 12 to 20 hours after inoculation into ES medium, separated into individual cells, and only the surviving cells were selected for use in the production of chimeric mice.

Selected cells are injected into the blastocyst of the blastocyst to prepare chimeric mice. At this time, blastocysts were bred from normal mouse females and males such as C57BL / 6 weeks, and then 3.5 P.C. The females were killed by cervical dislocation, the uterus was removed, the distal end of the uterus was excised with scissors, and a 1 ml syringe was used for injection solution [(20 mM HEPES, 10% fetal bovine serum (Hyclone Co.), 0.1 mM 2-mercapto). Ethanol, DEME (Gibco Co.)] was perfused twice with 1 ml, and blastocysts were separated using a microglass tube under a dissecting microscope, and the resulting blastocysts were placed in a drop of injection solution previously dropped on a 35 mm dish. The selected embryonic hepatocytes are introduced into the embryos, and the isolated embryos are held by a microinjector (Zeiss Corporation) and then held by a pipette. The direction of the inner cell mass (negative pressure) is taken, the inhalation of 10 to 15 embryonic stem cells into the injection pipette, and the injection pipette is inserted into the blastocyst of the blastocyst and given positive pressure to inject the cells into the blastocyst.

Embryonic stem cells injected embryos were incubated for 1 to 2 hours at 37 ° C., and then mated with males subjected to vas deferens before 2.5 P.C. The chimera mice are transplanted into the womb of the surrogate, surrogate mother mice. The surrogate mouse may be a lineage that manages the young mice well, such as FvB / N (Jacson laboratory, USA). As a transplant method. After the surrogate mother was anesthetized with 1 mg / kg of Avertin, the abdomen was resected about 1 cm, the upper part of the uterus was picked up with forceps, and pulled out of the body by about 2 cm. Embryos are injected into a microglass tube, the inner peritoneal membrane is sewn with two stitches, and the outer skin is sealed with a medical clip. The mice generated in this way become chimeric mice in which cells derived from embryonic stem cells and cells derived from blastocysts are mixed when about 19 days have elapsed.

In chimeric mice, cells derived from embryonic stem cells (J1) and cells derived from blastocysts (C57BL / 6) can be distinguished by phenotypes such as hair color. For example, embryonic stem cells have brown hair genes. The cells of the blastocyst carry a black hair gene. The higher the occupancy of brown color in the black background, the more chimeric mice are judged, and the higher chimeric male chimeric mice are bred with normal mice such as C57BL / 6 to obtain progeny. All of these offspring have brown hairs, 50% of which are heterozygous mice with a genotype of PLCβ1 ^+/- , which can be identified by conventional methods such as PCR.

The heterozygous mouse females and males are crossed to obtain variant mice of the present invention having a genotype of PLCβ1 ^{− / −} .

Variant mice of the present invention, most of the lethal and surviving 3 weeks after birth due to epilepsy, so even a small number of surviving mice, fertility is not obtained, the heterozygous mice obtained by crossing the male and male, or PLCβ1 ^{+ /} - Chimeric mice, heterozygous mice, and homozygous variant mice can be easily obtained from embryonic stem cells of a heterozygous heterozygous genome in this manner. Accordingly, the present inventors deposited the embryonic stem cell line β1 +/- J1-ES of a heterozygote having a genotype of PLCβ1 ^{+/- to} the gene bank affiliated with the Biotechnology Research Institute on February 23, 1998, with No. KCTC 0437BP.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1: Construction of a PLCβ1 Hit Vector

The cDNA of the rat PLCβ1 gene (obtained from Prof. Seopangil, Pohang University of Science and Technology) was digested with BglII and AccI to obtain a 0.2kb BglII / AccI fragment, which was then used as a probe to hybridize with the mouse genomic DNA library to make a genomic clone. Phage 9.1 was obtained, and a 7.6 kb BamHI fragment was obtained from this clone. This fragment contains base sequences corresponding to amino acid sequences 50-82 of the PLCβ1 protein (part of the PH domain). This fragment was inserted into the BamHI cleavage site of the plasmid pSK (Stratagene) to prepare plasmid pPLC-B7.6.

On the other hand, the plasmid pWH11 (Professor Shin Hee-sup Professor, Department of Biotechnology, POSTECH) was cut with BamHI to obtain a PGK-neo cassette, and treated with Klenow and inserted into the SmaI cleavage site of plasmid pSKSV40pA (Professor Hee-Sup Shin, Laboratory of Biotechnology, POSTECH). It was. The PGK-neo cassette and the SV40 poly (A) sequence were digested with NotI and BamHI and then treated with Klenow and inserted into the BglII cleavage site of the 7.6 kb BamHI fragment to prepare plasmid pDS-4. The BglII cleavage site is located in the exon of the PLCβ1 gene, and the 7.6kb fragment is divided into 2.8kb and 4.8kb fragments. Therefore, in the prepared plasmid pDS-4, the PLCβ1 gene is destroyed due to the insertion of the PGK-neo cassette and the SV 40 poly (A), and a 2.8 kb PLCβ1 gene homolog, PGK-neo cassette, and the SV 40 poly (A) sequence. And 4.8 kb of PLCβ1 gene homologous sequence.

Plasmid pPNTs containing thymidine kinase gene cassettes (Professor Shin Hee-seop, Laboratory of Biotechnology, Pohang University of Science and Technology) were digested with EcoRI and treated with Klenow to obtain sections. This fragment was connected to a 8.2 kb fragment obtained by sequentially treating plasmid pDS-4 with ClaI, Klenow, and NotI with a ligase to prepare a hit vector pDS-5 of PLCβ1. At this time, ClaI is located 3 'of PLCβ1 gene. Therefore, the constructed hit vector pDS-5 has a structure in which the 2.8 kb homologous fragment is attached to the 5 'side of the PGKneo cassette, and the 4.8 kb homologous fragment and the thymidine kinase are sequentially attached to the 3' side.

Example 2: Preparation of Embryonic Stem Cells

Embryonic stem cell line J1 (available from MIT's R. Jeananisch) was treated with ES medium [15% fetal bovine serum (Hyclone Co.), 1x penicillin-streptomycin (Gibco Co.), 1x non-essential amino acid (Gibco Co.) , 0.1mM 2-mercaptoethanol, DMEM medium (Gibco Co.)] and incubated for 2 to 3 days at 37 ℃ after the germ cell group was added to 1mM EDTA solution containing 0.25% trypsin to separate into single cells It was.

Example 3 Introduction of the Hit Vector

2 x 10 ⁷ isolated single embryonic stem cells and 25 µg of the hit vector prepared in Example 1 were subjected to electroporation under the conditions at 270V / 500 ㎌. Embryonic stem cells into which the hit vector was introduced were selected in a medium containing 0.3 mg / ml G418 and 2 μM of liver cyclovir. These embryonic stem cell clones were treated with trypsin at 12 to 20 hours after inoculation into ES medium and separated into individual cells, and only the surviving cells were selected and used for the production of chimeric mice.

Genomic DNA was extracted from the isolated clones, digested with BamHI and subjected to Southern blot using a 0.2 kb BglII / AccI fragment used in Example 1 as a probe. As a result, only one band of 7.6kb size was observed in normal cells that were not hit, whereas two bands of 4.8kb and 7.6kb were observed in the hit clone. PLCβ1 gene was hit in the clone isolated from this, it can be confirmed that it has a genotype of PLCβ1 ^{+ / +} . The isolated embryonic stem cells were treated with trypsin 24 hours after inoculation, and then only the surviving cells were used for microinjection.

Example 4: Preparation of Chimeric Mice

Females and males of C57BL / 6 week mice were bred and examined for mating. After mating, 3.5 P.C. females were killed by cervical dislocation and the uterus was extracted. Excision of the distal uterine section with scissors and 1 ml injection solution ((20 mM HEPES, 10% Fetal Serum (Hyclone Co.), 0.1 mM 2-mercaptoethanol, DEME (Gibco Co.)] using a 1 ml syringe After perfusion, the embryos were separated using a microglass tube under a dissecting microscope The separated embryos were transferred to a drop of injection solution previously dropped on a 35 mm dish. Using a holding pipette to hold the inner cell mass of the embryos at negative pressure, inhale 10-15 embryonic stem cells into which the hit vector was introduced into the injection pipette, and then insert the injection pipette. The embryos were inserted into the blastocyst and injected under positive pressure into the blastocyst, resulting in a chimeric mouse with a mixture of embryonic stem cell-derived cells and blastocyst-derived cells.

Example 5: Preparation of Heterozygous Mice

Male chimeric mice with a high brown share of black background among the chimeric mice were selected and crossed with C57BL / 6 mice to obtain progeny. In order to select heterozygous mice having a genotype of PLCβ1 ^+/− , PCR was performed as follows.

The tails of the progeny mice were cut out to 1.5 cm and then placed in 0.4 ml of lysis buffer [100 mM Tris (pH 8.0), 5 mM EDTA, 200 mM sodium chloride and 0.2% SDS], where 10 mg / ml protease K solution 10 After addition of μl it was treated for 12 hours at 55 ℃. 150 μl of 5M potassium acetate was added thereto, shaken and mixed, and the mixture was left at 4 ° C. for 30 minutes. Then, 0.4 ml of chloroform was added thereto, shaken, and centrifuged at 15,000 rpm. 0.5 ml of the supernatant was added to 1 ml of ethanol to precipitate genomic DNA, which was then washed with 70% ethanol, dried and dissolved in 50 µl of distilled water.

PCR was performed using 1 pL of this genomic DNA using 10 pmol of the primer of SEQ ID NO: 1 (F1-PLC), the primer of SEQ ID NO: 2 (B1-PLC), and the primer of SEQ ID NO: 3 (22-PGK), respectively.

5'-CAAGTTAAGTCCGGCAAACACC-3 '

5'-ACCTTGGGAGCTTTGGCGTG-3 '

5'-CTGACTAGGGGAGGAGTAGAAG-3 '

In this case, primers F1-PLC and B1-PLC amplify the region of 180 bp size of the normal PLCβ1 gene, and primers F1-PLC and 22-PGK are designed to amplify the 290 bp size region of the hit PLCβ1 gene. Normal mice and heterozygous mice were used as controls.

As a condition of PCR, the reaction was repeated 42 times for 1 minute at 94 ° C, 1 minute at 58 ° C, and 1 minute at 72 ° C. PCR products were observed by electrophoresis on 1.5% agarose gel and stained with ethidium bromide.

From the results, mice showing two bands of 180 bp size and 290 bp size were selected as heterozygous mice having a genotype of PLCβ1 ^+/- .

Example 6: Preparation of homozygous variant mice

Heterozygous Mice Prepared in Example 5 Females and males were crossed to obtain homozygous variant mice having a genotype of PLCβ1 ^{− / −} . Embryonic stem cell line PLCβ1 +/- J1-ES obtained from blastocysts generated from crossed fertilized eggs was deposited with KCTC 0437BP, February 23, 1998, to the Gene Technology Bank.

Southern blot, PCR, and Northern blot were performed to confirm the genotype of the prepared mutant mice, and Western blot was performed to confirm whether the hit gene was expressed in the hit mice.

(1) Southern blot

Genomic DNA was isolated from the tail tissues of variant mice in the same manner as in Example 5, and then digested with EcoRI. This genomic DNA was subjected to Southern blot using a 2.0 kb BamHI / EcoRI fragment obtained from phage 9.1 prepared in Example 1 as a probe. The control group was a normal mouse having a genotype of PLCβ1 ^{+ / +} and a heterozygous mouse having a genotype of PLCβ1 ^+/- .

The result is shown in FIG. In Figure 2, the first and second rows of control mice, the third row and the fourth row is another control heterozygous mouse, the fifth row homozygous variant mice, the sixth row of the heterozygous mice control Genomic DNA, where the 5.8 kb band is from the normal PLCβ1 gene and the 5.0 kb band is from the targeted PLCβ1 gene. As shown in Figure 2, the prepared mutant mice showed only 5.0 kb band, from which it can be confirmed that it has a genotype of PLCβ1 ^-/- .

(2) PCR

After genomic DNA was isolated from the tail tissue of the mutant mice in the same manner as in Example 5, PCR was performed on the same primer as in Example 5 with respect to 1 µl of this genomic DNA.

The result is shown in FIG. 3. In Figure 3, column M is Φ174 HaeHIII standard molecular weight label (NEB) and rows 1 and 2 are control mice, rows 3 and 4 are heterozygous mice, and control columns are homozygous variants. Type mouse, column 6 shows genomic DNA of heterozygous mice as controls, the 180 bp band is from the normal PLCβ1 gene and the 290 bp band is from the targeted PLCβ1 gene. As shown in Figure 3, the mutant mice show only 290 bp it can be confirmed that the genotype of PLCβ1 ^-/- .

(3) Northern Blot

The mutant mice were killed by cervical dislocation and the skulls were excised with scissors. The spinal cord and optic nerve fibers were cut out with forceps, and the brain was separated from the skull and stored in dry ice.

Total RNA from brain tissue was isolated using a Trizol kit (MRC) and hybridized to the brain RNA using the cDNA of the rat PLCβ1 gene as a probe. The control group was used brain RNA of normal mice.

The result is shown in FIG. 4. In FIG. 4, the first row is RNA of a normal mouse as a control, and the second row is brain tissue RNA of a variant mouse. As shown in Figure 4, the variant mice can be seen that the mRNA of PLCβ1 gene is significantly reduced.

(4) Western Blot

The cerebrum or cerebellum of the stored variant mice were treated with saline I (0.32 M sucrose, 10 mM HEPES, pH 7.4) to which protein inhibitors (PMSF 0.2 ml, 2 μM leupeptin, aprotinin 1 μg / ml) were added. ) And crushed in glass-Teflon. The lysate was centrifuged at 8,000 g at 4 ° C., and then the precipitated cell membrane fractions were dissolved in physiological saline II (0.1 M sodium phosphate buffer, pH 7.4) and precipitated again with ethanol. The precipitate was dissolved in physiological saline III (1% SDS, 5 mM EDTA, 10 mM Tris-HCl (pH 8.3), 10% sucrose) and the protein content was quantified according to the Bradford method. 0.2 mg of protein was electrophoresed on a 12% acrylamide gel and transferred to the nitrocellulose membrane in transfer buffer (25 mM Tris-HCl, pH 8.3), 192 mM glycine, and 20% methanol). The membrane was protected with 5% skim milk and combined with an antibody to PLCβ1 (UBI Co.), followed by 1% BSA-TBST solution (50 mM Tris-Cl (pH 7.5), 0.5M NaCl, 0.2% sodium chloride, 0.2% Tween). 20). Subsequently, color reaction was performed using an ECL kit (Amercham) as a substrate using an anti-rabbit IgG antibody (Serotech) bound to peroxidase. The control group was used brain RNA of normal mice.

The result is shown in FIG. In Figure 5, C is a PLCβ1 protein purified from rats, the first column is the cerebral protein of the normal mouse control, the second column is the cerebral protein of the variant mouse, the third column is the cerebellar protein of the normal mouse control , Column 4 is the cerebellar protein of variant mice. As shown in FIG. 5, the purified PLCβ1 protein was not produced at all in the mutant mice.

Example 7: Phenotypic analysis of variant mice

In order to analyze the phenotype of the mutant mice prepared in Example 6, visual observation and sensitivity analysis of the epileptic-inducing substance were performed.

(1) visual observation

36 mutant mice were bred at 25 ° C for 12 hours under dark conditions and 12 hours under light conditions to measure survival. Normal mice were used as a control. The results are shown in Figure 6, where □ is a control mouse, ■ is a variant mouse. As shown in Figure 6, the mutant mice are born normally but begins to die at 20 days of age and the survival rate is drastically decreased with time.

In addition, the video was taken while breeding mutant mice and visually observed through this video. The cause of death was repeated epilepsy. That is, as shown in Figure 7, the entire body showed tonic extension (tonic extension) and the tail and legs showed systemic epilepsy indicating the tonic-clonic spasms.

(2) Sensitivity analysis of epileptics

In normal mice, 50 mg / ml of kininic acid or 10 mg / ml of quinine acid, which is less than the amount required to induce epilepsy, was administered to the abdominal cavity of three-week-old mutant mice, and the seizure behavior was observed. . At this time, a heterozygous mouse was used as a control.

The results are shown in Figures 8a to 8d. Here, Figure 8a is a mutant mouse administered with the chininic acid, Figure 8b is a control mouse administered with the kinanic acid, Figure 9c is a mutant mouse administered with the metrazol, Figure 8d is a control mouse administered with the metrazol , The black box indicates systemic epilepsy showing hepatic, tonic-clonic or tonic expansion, the box with crossed parallel lines indicates no temporary stiffness or epilepsy of the legs or tail, and the gray box shows repeated scratches, Stereotypes such as rotation, hindlimb, etc., and square patterning represent post-letal periods including hypokinasia, recovery of sleep or physical control, and the time interval is shown in FIG. 8A Equivalent to 2 minutes per box in and 8b, 20 seconds per box in white boxes or crossed parallel lines, and 200 seconds per box of black boxes or dot marks in FIGS. 8C and 8D. Suffer As shown in Figure 8a to 8d, the mutant mice can be seen that the sensitivity to chiinic acid and metrazole.

Example 8: Immunohistochemical Analysis

Mutant mice with spontaneous epilepsy were anesthetized with 1 mg / kg Avertin and 4% paraformaldehyde was perfused to the heart. Brains were extracted from the mice, encapsulated in paraffin wax, and cut into 50 μm thickness using vibratome. The fragment containing the hippocampus in this section was reacted for 6 hours at 4 ° C. using a somatostatin polyclonal antibody as the primary antigen, stained using a peroxidase kit (Vector), and immunohistochemical analysis ( immunohistochemical analysis) was performed. Normal mice were used as a control.

The results are shown in Figures 9A-9F, where 9a, 9c and 9d are hippocampus of normal mice, 9b, 9e and 9f are hippocampus of mutant mice, and the actual size of the bars in 9a and 9b is 200 μm, 9c The actual size of the bar from 9 to 9f is 40 μm. As shown in FIGS. 9A-9F, somatostatin-containing intermediate neurons were selectively lost at the gate of the hippocampus. This suggests that epilepsy in mutant mice is due to overexcitement of the hippocampus. In addition, since this phenomenon is also observed in human epilepsy, it can be seen that the epilepsy of the mutant mice of the present invention is similar to that in humans, and the site where seizures mainly occur is the hippocampal area where the somatostatin-containing intermediate neurons are necrotic.

Example 9 Hydrolysis of Phosphoinositide

Phosphoinositiated hydrolysis experiments were performed on a cut 0.35 mm hippocampus, 0.35 cm x 0.35 cm temporal cortex and 0.4 cm x 0.35 cm cerebellum (Beriddge et al., Biochem. J., 212, 473-482 (1983)) was modified as follows. In other words, 20 μl of a gravity-packed brain slice was prepared using Krebs bicarbonate buffer solution containing 1.3 μl of 2- ³ [H] -myo-inositol (1.3 mM calcium chloride solution in 95% oxygen / 5% carbon dioxide gas). Saturated under) to 0.25 ml and reacted for 1 hour at 33 ° C. for radiolabeling, then treated with 10 mM lithium chloride and 10 mM myo-inositol for 20 minutes at 33 ° C., followed by 1 mM carbacol (CCh), 100 μM 1 Amino-1,3-cyclopentanedicarboxylic acid (ACPD) or 2 mM 1- (3-chlorophenyl) piperazine (mCPP) was reacted at 37 ° C. for 40 minutes. 1 ml of a mixture of chloroform and methanol (mixing ratio of 1: 2) was added to the reaction solution to terminate the reaction, and 0.6 ml of a mixture of chloroform and water (mixing ratio of 1: 1) was added and extracted. Total inositol polyphosphate in the aqueous layer was quantified after separation using an AGIX-8 column (Bio-rad). Phosphoinositide hydrolysis induced by CCh, ACPD or mCPP was expressed as a percentage of control (base) cpm untreated with reagents.

The result is shown in FIG. As shown in FIG. 10, in the hippocampus of mutant mice, the hydrolysis of phosphoinositide induced by CCh was significantly delayed, whereas the hydrolysis of phosphoinositide induced by ACPD was significantly increased and increased in mCPP. The hydrolysis of the phosphoinositide induced by does not change at all. The delay in hydrolysis is also the same in the temporal cortex and cerebellum of variant mice. These results indicate that CCh is a nonselective agonist for the muscarinic acetylcholine receptor, ACPD is a nonselective agonist for the metabotropic glutamic acid receptor, and mCPP is a serotonin (5-HT) type-2 receptor. From the fact that it is a non-selective agonist for mutant mice, it can be seen that the signal transmission by muscarinic acetylcholine at the hippocampus is selectively damaged and blocked.

It was first discovered in the present invention that PLCβ1 protein is involved in the signaling system by being linked to muscarinic acetylcholine receptors in the temporal cortex and hippocampus.

Mutant mice of the present invention have a genetic epilepsy due to a deletion of the PLCβ1 gene according to the gene targeting method, and is useful as an experimental animal model for understanding epilepsy mechanisms and developing epileptic therapeutic agents.

Claims (4)

An epilepsy-induced mus muculus mutant mouse, characterized in that it comprises a homozygous variant mutant PLCβ1 gene selected from the base sequence from the PLCβ1 gene initiation site to the PH domain. The method of claim 1, Mousse muculus mutant mouse, wherein the site is the PH domain. The method of claim 2, Moose muculus mutant mice derived from embryonic stem cells (Accession No. KCTC 0437BP). (1) introducing a hit vector for a site selected from the nucleotide sequence of the PLCβ1 gene corresponding to the amino acid sequence from the N-terminus to the PH domain of PLCβ1 into embryonic stem cells, and (2) introducing the embryonic stem cells into blastocysts. Obtaining chimeric mice by injecting into the lumen, (3) crossing the chimeric mice to obtain heterozygous mice, and (4) crossing the heterozygous mice to obtain homozygous variant mice Method for producing a variant mouse of claim.