KR20090081232A - PLCbeta4 MUTANT MICE AS A MODEL FOR TESTING ANXIETY DISORDER DRUGS - Google Patents

Abstract

Provided are a method or a use of using a PLC beta4 knock-out animal as an anxiety disorder animal model, and a method of screening anxiety disorder-preventing and treating agents using the animal. A method of using a PLC beta4 knock-out animal as an anxiety disorder animal model comprises using an animal in which a PLC beta4(phospholipase beta4) gene representing a lack in the memory disappearing capability is knocked-out as an anxiety disorder animal model. A use of using a PLC beta4 knock-out animal as an anxiety disorder animal model comprises using an animal in which a PLC beta4(phospholipase beta4) gene representing a lack in the memory disappearing capability is knocked-out as an anxiety disorder animal model. A method of screening anxiety disorder-preventing and treating agents comprises the steps of: injecting candidate materials of the anxiety disorder-preventing and treating agents into an animal in which a PLC beta4(phospholipase beta4) gene is knocked-out; testing the memory disappearing capability of the animal; and sorting the candidate material that has attentively recovered the memory disappearing capability as compared with a control group into which the candidate materials have not been injected.

Description

PLββ4 mutant mice as a model for testing anxiety disorder drugs

The present invention relates to the use of PLCβ4 knock-out animals, and more particularly, to a method or use of PLCβ4 knock-out animals as an anxiety disorder animal model, and to prevent and treat anxiety disorders using the animals. It relates to a screening method of.

Anxiety disorders include phobic disorders, generalized anxiety disorders, obsessive compulsive disorders, post-traumatic stress disorders, somatoform disorders, and dissociative disorders ( dissociative disorders or factitious disorders. A fear disorder is a disorder in which a person feels irrational fear in a particular subject or situation and continually avoids it because of social phobia, acrophogia, claustrophobia, or antiexpectation. There are many types, depending on their targets. A general anxiety disorder is one in which a person feels widespread and persistent anxiety in everyday situations, and an obsessive compulsive disorder usually involves a certain amount of compulsive thoughts or actions, regardless of his will. Although obsessive-compulsive symptoms can interfere with your life or bother you, you will be diagnosed with OCD. Usually they appear to be a characteristic of compulsive personalities such as accuracy, perfection, and principleism. Post-traumatic stress disorder can be re-experienced by experiencing extremely traumatic events (war, plane accidents, rapes, Sampoong Department Store accidents, etc.) that people cannot experience in general, and then repeatedly thinking about such terrible accidents or appearing in dreams. Refers to disorders that are insensitive to the outside and accompanied by symptoms of the autonomic nervous system. Somatoform disorders are cases in which physical symptoms are suspected of physical illness, but in reality there are no physical abnormalities and physical symptoms are caused by psychological conflicts or factors. Psychological conflicts or factors often cause physical symptoms or illnesses, which are called psychophysiological disorders. The expression of psychological conflicts as physical symptoms is generally thought to occur on an unconscious level. In other words, it is different from malingering, in which the patient is consciously and deliberately expresses physical symptoms. Dissociative disorders are rare, as in the case of Dr. Jekyll and Hyde, where suddenly part of the mental function is temporarily lost.

The dorsal medial nucleus (mediodorsal thalamus, MD) is part of the limbic system and is connected to the frontal lobe, the amygdala and the hippocampus, which are related to emotion. However, the mechanism by which MD is involved in the act of emotion is not well known. Phospholipase β4 (PLβ4) is highly expressed in MD but hardly expressed in the frontal or amygdala, which are important for fear disappearance (Vertes RP. Neuroscience . 2006 Sep 29; 142 (1): 1-20; Oyoshi T, et al ., J Neurosci . 1996 Sep 15; 16 (18): 5812-29; Kuroda M, et al ., Prog Neurobiol . 1998 Mar; 54 (4): 417-58; Nakamura M, et al ., Eur J Neurosci . 2004 Dec; 20 (11): 2929-44).

Accordingly, the present inventors have completed the present invention by confirming that the PLCβ4 knock-out mouse can be used as an animal model for anxiety disorder showing a severe lack of memory extinction ability.

It is an object of the present invention to provide a PLCβ4 knock-out animal model that can be used for screening drugs for the prevention or treatment of anxiety disorders by indicating a severe lack of memory extinction capacity.

In order to achieve the above object, the present invention provides a method of using an animal model knock-out of the phospholipase β4 gene showing a lack of memory extinction ability as an anxiety disorder animal model.

In addition, the present invention provides a use of an animal in which an phospholipase β4 gene showing a lack of memory extinction ability is knocked out as an anxiety disorder animal model.

In addition, the present invention provides a method for screening an agent for preventing and treating psychotic disorders using an animal in which the phospholipase β4 gene is knocked out.

The PLCβ4 knock-out mouse of the present invention has a normal learning ability, long-term memory and activity ability, but the memory extinction ability is remarkably lowered, and thus, as an effective animal model for deciding the causes of various anxiety disorders and screening preventive and therapeutic agents. It can be usefully used.

Hereinafter, the present invention will be described in detail.

The present invention provides a method and use of a knock-out animal having a phospholipase β4 gene showing a lack of memory extinction ability as an anxiety disorder animal model, and the phospholipase β4 ( The present invention provides a method for screening an agent for preventing and treating psychotic disorders using an animal in which the phospholipaseβ4) gene is knocked out.

The method is

1) administering a candidate for preventing and treating anxiety disorder to an animal knocked out of the phospholipase β4 gene;

2) after the administration of the candidate substance of step 1), performing a memory extinction ability test of the animal; And

3) selecting a candidate material that significantly restored the memory extinction ability compared with the control group not administered the candidate material.

Anxiety disorders in step 1) include phobic disorders, generalized anxiety disorders, obsessive compulsive disorders, post-traumatic stress disorders, and somatoform disorders. ), Dissociative disorder and artificial disorder (factitious disorder) is preferably selected from the group, but is not particularly limited thereto.

The animal of step 1) may be a mammal, preferably a mouse, a rat, a pig or a monkey, and the like, and most preferably a mouse generated from a fertilized egg (accession number: KCTC 11247BP) deposited by the present inventors. It is not limited.

Candidates of step 1) include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts or plasma, and these compounds may be novel or widely known compounds. . Such a candidate substance may form a salt. Salts of candidate substances include salts such as physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids, etc.), and among these, physiologically acceptable acid addition salts are preferable. Such salts include, for example, salts of inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid). , Malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

As a method of administering the candidate substance as described above, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration can be appropriately selected according to the symptoms of the target animal, the nature of the candidate substance, and the like. In addition, the dosage amount of a candidate substance can be suitably selected according to the administration method, the nature of a candidate substance, etc.

The memory extinction ability test of step 2) means that the test is performed by periodically exposing the environment related to the conditioning after 1 hour to 60 hours after the conditioning reaction, and then measuring the freezing reaction. In addition, the cycle is preferably performed 20 to 30 times the conditioning reaction for 15 seconds to 50 seconds at intervals of 3 seconds to 180 seconds, but is not particularly limited thereto.

A knock-out mouse (PLCβ4) that lacks the phospholipase β4 (PLCβ4) gene, which is expressed in the medial nucleus of the thalamus (MD) but rarely expressed in the frontal or amygdala, which are important for fear extinction. -Out mice), the inventors first prepared a vector lacking the PLCβ4 gene and introduced it into embryonic stem cells of the mouse. Next, embryonic stem cell clones in which the PLCβ4 gene deficiency was confirmed were cultured, injected into the blastocyst of the blastocyst, crossed with males subjected to vas deferens, and transplanted into the uterus of surrogate mice to induce the development of chimeric mice. The chimeric mice were bred with normal mice to obtain heterozygote mice having the PLCβ4 +/- genotype, and again the females and males of the heterozygous mouse were transgenic mice having the PLCβ4-/-genotype (PLCβ4). Knock-out mice). In the present invention, the PLCβ4 knock-out mouse prepared as described above was used as an animal model of anxiety disorder, and the fertilized egg of PLCβ4 knock-out mouse was deposited in KCTC (Korean Collection for Type Cultures) on December 6, 2007 ( Accession number: KCTC 11247BP).

The present inventors measured the freezing reaction of the PLCβ4 knock-out mouse in a specific extinction chamber in which external sound was blocked by learning, long-term memory, activity, and memory extinction. Tested whether it can be used as.

In the ability test in classical fear conditioning experiments, the PLCβ4 knock-out mouse was the same as a normal mouse, and as the attempts at conditioning on specific sounds increased, the percentage of freezing reactions also increased (see Figure 1). It can be seen that the learning ability of PLCβ4 knock-out mice is normal.

Long-term memory testing of PLCβ4 knock-out mice showed that long-term memory was also normal by showing a percentage of freezing response similar to that of normal mice for certain conditioned sounds (see FIGS. 2 and 3).

In the activity ability test of the PLCβ4 knock-out mouse, the distance of the mouse movement in the specific extinction chamber in which the external sound was blocked was measured. As a result, there was no difference as compared with the normal mouse (see FIG. 4). ), The activity capacity of PLCβ4 knock-out mice does not affect freezing.

In 24 hours after the conditional learning of a specific sound in the memory ability test, the normal mouse disappeared in 20 attempts, while the fear memory for the sound disappeared, whereas in the PLCβ4 knock-out mouse (Fig. 5) After 24 hours thereafter, normal mice disappeared in PLCβ4 knock-out mice, whereas the fear memory for a particular sound disappeared in 6 trials (see FIG. 6). As a result, the PLCβ4 knock-out mouse was found to have abnormal fear memory disappearance for a particular sound.

In general, patients with anxiety disorders are treated by repeated exposure of the patient to a specific, fearless, conditioned condition. Therefore, the memory annihilation experiments of the horror conditioning used in the present invention have been used for a long time and have been used continuously since Pavlov (1927) (Myers KM and Davis M. Neuron . 2002 Nov 14). 36 (4): 567-84; Milad MR and Quirk GJ. Nature. 2002 Nov 7; 420 (6911): 70-4; Marsicano G, et al ., Nature . 2002 Aug 1; 418 (6897): 530-4).

Therefore, as discussed above, PLCβ4 knock-out mice have normal learning ability, long-term memory, and activity ability, but their fear memory extinction ability is significantly lowered, and thus can be usefully used as an animal model of anxiety disorder.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following Examples are only for illustrating the present invention and do not limit the present invention.

< Example  1> Phospholipase β4 ( Phospholipase β4) gene knock-out ( knock - out ) mouse( PLCβ4 knock-out mouse)  Produce

<1-1> PLC Preparation of β4 Gene Deletion Vectors

To isolate the PLCβ4 gene from the mouse genome, 226 to 369 sites from the start codon of the cDNA (SEQ ID NO: 1) of the PLCβ4 gene were isolated by RT-PCR and used as a probe. Hybridization reactions were performed on svJae mouse genomic DNA library (LamdaFixII, Stratagene Inc., USA). From this, genome clone phage having PLCβ4 gene was selected, and it was confirmed that the phage clone was PLCβ4 gene by restriction enzyme map, Southern blot analysis and sequencing.

To prepare a vector lacking the PLCβ4 gene, a portion of the X domain of the PLCβ4 protein was removed from the PLCβ4 gene clone and cloned into a PSK-plasmid vector (Stratagene Inc., USA). A thymidine kinase gene cassette and a negative selection marker were inserted at the 3 'homologous end of the hit vector including a part of the PLCβ4 gene deleted.

<1-2> cell culture

J1 embroinic liver cells were used as a cell line for transducing the hit vector prepared in Example <1-1>. J1 embryonic stem cells (received from R. Jeanisch, Massachusetts Institute of Technology, USA) were placed in DMEM medium (Gibco Co., USA) in 15% fetal bovine serum, Hyclone Co., USA, 1 × penicillin -Inoculated in ES medium with phenicillin-streptomycin, 1 × non-essential amino acid (Gibco Co., USA), 0.1 mM 2-mercaptoethanol and then at 37 ° C. for 2-3 days Incubated. The germ cell group obtained from the culture was treated with 1 mM EDTA solution containing 0.25% trypsin to isolate single cells.

<1-3> into cells PLC Introduction of β4 Gene Deletion Vectors

Electroporation was performed to transfect the hit vector prepared in Example <1-1> to embryonic stem cells separated into single cells in Example <1-2>. Specifically, 25 μg of the hit vector DNA prepared in Example <1-1> was added to the embryonic hepatocytes diluted to a concentration of 2 × 10 ⁷ cells / ml and mixed, followed by electric shock at 270 V / 500 μF. Was added. The embryonic hepatocytes were cultured in ES medium containing 0.3 mg / ml G418 and 2 μM hepatocyclovir for 5 to 7 days, and the PLCβ4 gene in embryonic hepatocytes was correctly hit by a hit vector by homologous recombination. Embryonic stem cell clones were selected and maintained by Southern blot.

<1-4> PLC Preparation of β4 +/− Mice

To prepare chimeric mice with genotype PLCβ4 +/−, fertilized embryonic cells were microinjected with the embryonic hepatocyte clones selected in Example 1-1 above.

Specifically, females and males of C57BL / 6J mice (Jackson Laboratory, USA) were crossed, and 3.5 days (3.5 p.c.) of females were sacrificed by cervical dislocation. After removal of the uterus from the sacrificed females and excision of the terminal part of the uterus with scissors, 1 ml injection solution containing 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM was added using a 1 ml syringe. Perfusion. The blastocyst was separated from the uterine tissue under a dissecting microscope using a micro glass tube, and the separated blastocyst was transferred to a drop of injection solution previously dropped on a 35 mm Petri dish and used for the following introduction process.

In order to introduce the embryonic stem cell clones selected in Example <1-3> to the isolated embryos as described above, internal cell masses of the embryos were held by a holding pipette using a microinjector (Zeiss Inc., USA). An injection pipette with 10-15 embryonic stem cell clones inhaled with negative pressure in the direction of (inner cell mass) was inserted into the blastocyst of the blastocyst and injected into the blastocyst of the blastocyst under positive pressure. The clone-implanted blastocysts were mated with a vas deferens male, and then transplanted into the uterus of 2.5 pcs of fertility surrogate mice to form heterologous cell hybrids formed from embryonic stem cell clones (J1) and blastocysts of C57BL / 6J mice. Induction of in chimeric mice was induced. At this time, uterine transplantation is excised the abdomen of the surrogate mother anesthetized with avertine (1 mg / kg per body weight) by about 1 cm; Pick the upper uterus with tweezers and pull it out of the body about 2 cm; Puncturing the uterus with a needle to inject the blastocyst into the microglass tube through the hole; The inner peritoneal membrane was sewn with two stitches, and the outer skin was sealed with a medical clip. As described above, the embryos injected with embryonic stem cells were transplanted into the uterus of the surrogate mouse and cultured for about 19 days, whereby the cells derived from embryonic stem cells and the cells from the embryos were fused to have a genotype of PLCβ4 +/- in the genome. Mice were obtained.

<1-5> PLC Preparation of β4 Knock-out Mice

The chimeric mice were maintained at least 20 crossings with C57BL / 6J and 129sv mice, respectively, and the resulting C57BL / 6J-PLCβ4 + // and 129sv-PLCβ4 +/- mice were crossed to PLCβ4 + / + and PLCβ4- / at the F1 stage. Mice were prepared and used in the following behavioral experiments. Genotyping was performed using polymerase chain reaction (PCR). Primers were prepared using K1 (5'-CTCCACACTCTGCAACCTAC-3 '; SEQ ID NO: 2), K9 (5'-AGTTACTTCTGGATTTTCAGCC-3'; SEQ ID NO: 3) and PGK22 (5'-CTGACTAGGGGAGGAGTAGAAG-3 '; SEQ ID NO: 4). The reaction was carried out 40 times in 30 seconds at 30 캜, 30 seconds at 58 캜, and 30 seconds at 72 캜. The primer K1 and K9 was used as a primer pair for identifying genotypes of normal mice, and primers K1 and PFK22 were used as primer pairs for identifying genotypes of mutant mice. The PCR product thus obtained identified each band in 1.5% EtBr / agarose gel. (PLCβ4 + / +: 190 bp, PLCβ4 +/-: 250/190 bp and PLCβ4-/-: 250 bp)

< Example  2> Breeding and composition of mouse

The mice were bred in a SPF (specific pathogen free) environment with free water and feed under a 12-hour bright, 12-hour dark-illuminated circadian, and at a temperature of 22 ° C. and a humidity of 55%. The mice used in the experiment were all males and 6-18 animals were used. Statistical tests were performed using T-test and repeated two-way ANOVA.

< Example  3> PLC Investigation of Learning Ability in Classical Fear Conditions of β4 Knock-out Mice

 To investigate the learning ability of PLCβ4 knock-out mice, freezing reactions were measured.

In a specific conditioning chamber where external sounds are blocked, audible specific sounds (2900 Hz, 100 db) are heard for 30 seconds and the sole electrical stimulation (0.5 mA), which causes the freezing reaction, is heard. A stimulus was performed three times at 120-second intervals, giving one second but ending at the same time as a specific sound.

As a result, the freezing response was almost 0% in the first trial through the above learning, but as the condition became condition, the freezing response to the specific sound gradually increased in the second and third trials. And PLCβ4 knock-out mice (FIG. 1). That is, PLCβ4 knock-out mice had no difference in learning ability compared to normal mice.

< Example  4> PLC Investigation of Long-term Memory in β4 Knock-out Mice

To investigate the long-term memory of PLCβ4 knock-out mice, freezing reactions were measured.

In order to measure the memory capacity for a particular sound condition only in a box having an environment (sound, smell, illuminance, color, texture, etc.) completely different from the specific box that was conditioned in Example 3, 24 After the hour and 48 hours had elapsed, the freezing response was measured when one particular sound was given for 30 seconds.

As a result, PLCβ4 knock-out mice showed freezing reactions of 93.6 ± 0.8% [mean ± standard error of the mean (SEM), FIG. 2] and 95 ± 1.1% (FIG. 3). Similar to the out-out mice, the freezing response was 88.3 ± 2.8% (FIG. 2) and 84.4 ± 2.9% (FIG. 3). Therefore, PLCβ4 knock-out mice were found to have normal long-term memory for specific conditioned sounds.

< Example  5> PLC Investigation of the Activity Capacity of β4 Knock-out Mice

Since the degree of freezing response is correlated with the activity ability of the mouse, the moving distance which is widely used as the activity ability was measured.

After 24 hours, the total distance measured by the mouse for 10 minutes was measured in the same box as Example 4 after 24 hours.

As a result, the movement distance of the PLCβ4 knock-out mouse (2854.2 ± 150 cm) and the movement distance of the normal mouse (2394.3 ± 230.5 cm) were similar (Fig. 4). It was found that there is no.

< Example  6> PLC Investigation of Amnesia in β4 Knock-out Mice

Normal mice and PLCβ4 knock-out mice as in Example 3 After 24 hours of conditioned sound, a specific sound (30 seconds) was heard 20 times in the conditional sound memory test box (extinction day1). The specific sound and the specific sound interval were 5 seconds. 24 hours later, the sound was repeated six times (extinction day2).

As a result, the fear memory for sound disappeared with 20 trials in normal mice, while not significantly destroyed in PLCβ4 knock-out mice (FIG. 5). After 48 hours of conditioned sound, the freezing reaction was significantly higher in the normal mouse than in the normal mouse, whereas the fear memory for that particular sound disappeared in six trials. 6).

1 is a graph showing the learning ability of PLCβ4 knock-out mice in classical fear conditioning experiments:

    + / +: Normal mice; And

    -/-: PLCβ4 gene knock-out mouse.

FIG. 2 is a graph showing the long-term memory of PLCβ4 knock-out mice for specific conditioned sounds after 24 hours after conditioning:

    + / +: Normal mice; And

    -/-: PLCβ4 gene knock-out mouse.

FIG. 3 is a graph showing the long-term memory of PLCβ4 knock-out mice for specific conditioned sounds after 48 hours after conditioning:

    + / +: Normal mice; And

    -/-: PLCβ4 gene knock-out mouse.

4 is a graph measuring the moving distance of a PLCβ4 knock-out mouse in a memory test box for a specific sound:

    + / +: Normal mice; And

       -/-: PLCβ4 gene knock-out mouse.

FIG. 5 is a graph showing the results of an extinction training day1 test for specific conditional sounds of a PLCβ4 knock-out mouse in a memory test box for a specific sound:

     + / +: Normal mice; And

     -/-: PLCβ4 gene knock-out mouse.

FIG. 6 is a graph showing the recall of extinction day2 results after 24 hours of fear memory extinction learning for conditional specific sounds of PLCβ4 knock-out mice in a memory test box for specific sounds:

     + / +: Normal mice; And

     -/-: PLCβ4 gene knock-out mouse.

<110> Korea Institute of Science and Technology <120> PLC beta4 mutant mice as a model for testing anxiety disorder          drugs <130> 7p-06-12 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 3528 <212> DNA <213> Mus musculus <400> 1 atggccaaac cttacgaatt taactggcag aaggaagtgc cctctttctt gcaagaagga 60 gcagtttttg acagatacga agaggaatct tttgtgtttg agcccaactg cctcttcaaa 120 gtagatgaat tcggcttctt cctgacgtgg aagagtgaag gcaaggaagg acaagtgcta 180 gaatgttccc tcatcaacag tattcgccaa gcagccatac caaaggatcc caaaatcctg 240 gctgctctcg aagctgttgg aaaatctgaa aatgatctgg aagggcggat attgtgtgtc 300 tgcagcggca cggatctggt gaatatcggc ttcacttaca tggtggctga aaatccagaa 360 gtaactaagc aatgggtaga aggcctgaga tcgatcattc acaacttcag ggcaaacaac 420 gtcagtccga tgacatgcct caagaaacac tggatgaaac tggcctttct gaccaacaca 480 actggtaaaa tcccagtgag gagtatcact agaaccttcg catcagggaa aacagaaaag 540 gtgatctttc aagccctcaa ggaactaggt cttcccagtg gaaagaatga tgaaattgaa 600 cctgctgcat ttacttatga aaagttctat gaactgacac aaaagatttg tcctcggaca 660 gatatagaag atctttttaa aaaaatcaat ggagacaaaa ctgattattt aacggtagac 720 caattagtga gctttctaaa cgaacatcag cgagatcctc ggctgaatga aattttattc 780 ccattttatg atgctaaaag agcaatgcag atcattgaaa tgtatgagcc tgatgaagag 840 ctgaagaaaa aaggcctcat atccagtgat ggattctgca gatatctgat gtcagatgaa 900 aatgcccctg tcttcttaga tcgcttagaa ctttaccagg agatggacca cccgctggct 960 cattacttca tcagttcctc ccacaacacc tatctcactg gccggcaatt tggaggaaag 1020 tcttcagtgg aaatgtacag acaagttctc ctggctggtt gcaggtgtgt tgaacttgac 1080 tgttgggatg gaaaaggtga agatcaggaa ccgataataa ctcacggaaa agcaatgtgt 1140 acagacatcc tttttaagga tgtaatccag gccatcaagg aaacggcgtt tgtcacatca 1200 gaataccctg tcattctctc cttcgaaaac cactgcagca aatatcaaca gtacaagatg 1260 tccaagtatt gtgaagatct atttggggat ctcctgttga aacaagcact tgagtcgcat 1320 ccacttgaac caggaaggcc cttgccgtct cctaatgacc tcaaaagaaa aatactcatc 1380 aagaataaga ggctgaagcc tgaagttgaa aagaaacagc ttgaagcttt gaaaagcatg 1440 atggaagctg gagagtcagc cgccccagct agcatcttgg aagacgacaa tgaagaggaa 1500 atagaaagtg ctgatcaaga ggaagaagcc caccctgaat acaaatttgg aaatgaactt 1560 tctgccgatg actacagtca caaggaagcg gttgcaaaca gcgtcaagaa gggcctggtc 1620 accgtagagg atgagcaagc atggatggca tcttataaat acgtaggtgc taccacgaac 1680 atccatccgt acttgtccac gatgatcaac tatgcccagc ccgtgaagtt tcaaggtttc 1740 cacgtggctg aagagcgcaa tattcactat aacatgtctt cttttaacga gtcggttggc 1800 cttggctact tgaagacgca cgcgattgag tttgtaaatt acaataagcg acaaatgagc 1860 cgcatttacc ccaagggagg ccgagttgat tccagtaatt acatgcctca gattttctgg 1920 aacgctggtt gccagatggt ttcactgaac tatcaaaccc cagatttagc gatgcaattg 1980 aatcaaggaa aatttgagta taatggatca tgcgggtacc ttctcaagcc agatttcatg 2040 aggcggcctg atcggacatt tgaccccttc tctgaaaccc ctgtggacgg ggttattgca 2100 gccacgtgct cagtgcaggt tatatcaggg cagttcctct cagataagaa gatcgggaca 2160 tacgtggaag tcgatatgta cgggctgccc accgacacca tacggaaaga gttccgaacc 2220 cgcatggtta tgaacaatgg actcaaccca gtgtataatg aagaatcgtt tgtgtttcga 2280 aaggtgatcc tgcctgacct agctgtcctg agaatcgcag tctacgatga caacaacaag 2340 ctaattggcc agaggatcct tcctttggat ggtctccaag caggctaccg acacatctcc 2400 ctgagaaacg agggaaacaa accattatca ctgccaacaa ttttctgcaa tattgttctt 2460 aaaacatacg tgcctgatgg atttggagat attgtggatg ctttatccga tccaaagaaa 2520 tttctttcaa tcacagagaa gagagcagac caaatgagag caatgggcat tgaaactagt 2580 gacatagcag atgtgcccag tgacacttcc aaaaatgaca agaaaggcaa ggccaaccca 2640 gccaaagcga acgtgacccc tcagagcagc tctgagctca gaccaaccac cacagccgcc 2700 ctgggctctg gccaggaagc caagaaaggt attgaactta tccctcaagt gaggatagaa 2760 gatttaaagc aaatgaaggc ttacttgaag catttaaaga aacaacagaa ggagttaaac 2820 tctttaaaga agaaacatgc aaaggagcac agtaccatgc agaagttaca ctgcacacaa 2880 gttgacaaaa tcgtggccca gtatgacaaa gagaagtcga ctcatgagaa aatcctagag 2940 aaggcgatga agaagaaggg gggaagtaat tgtcttgaaa taaaaaaaga aacagaaatt 3000 aaaattcaga ccctgacaac ggatcacaaa tctaaggtca aagagattgt ggcccagcac 3060 acaaaggagt ggtcagaaat gatcaacact cacagtgcgg aggagcagga aatccgggat 3120 ctgcacctga gccagcagtg tgagctgctg agaaagctgc tcatcaatgc tcatgagcag 3180 cagacccagc agctgaaact ctcccatgac agggaaagca aggagatgag agcccatcag 3240 gctaagattt ctatggaaaa tagcaaggcc atcagtcaag ataaatctat caagaacaag 3300 gcagaacggg aaaggcgagt cagggagttg aacagcagca acactaagaa gttcctagaa 3360 gaaagaaaga gactggcgat gaagcagtca aaagaaatgg atcagttgaa aaaagtccag 3420 ctggagcacc tagaattcct agagaaacag aacgagcagg cgaaggagat gcagcagatg 3480 gtgaaattgg aagccgagat ggaccgcaga ccagcaacag tagtatga 3528 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer k1 <400> 2 ctccacactc tgcaacctac 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer k9 <400> 3 agttacttct ggattttcag cc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer PFK22 <400> 4 ctgactaggg gaggagtaga ag 22  

Claims (10)

A method of using a knock-out animal having a phospholipase β4 gene showing a lack of memory extinction as an animal model of anxiety disorder. Use of an animal model with an anxiety disorder knock-out of a phospholipase β4 gene showing a lack of memory extinction ability. 1) administering a candidate for preventing and treating anxiety disorder to an animal knocked out of the phospholipase β4 gene; 2) after the administration of the candidate substance of step 1), performing a memory extinction ability test of the animal; And 3) Screening method for preventing and treating anxiety disorders comprising selecting a candidate substance that significantly restored the memory extinction ability compared with the control group not administered the candidate substance. 4. The anxiety disorder of claim 3, wherein the anxiety disorder comprises a phobic disorder, a generalized anxiety disorder, an obsessive compulsive disorder, a post-traumatic stress disorder, or a body type disorder. somatoform disorder, dissociative disorder and factitious disorder. 4. The method of claim 3, wherein the animal of step 1) is a mammal. 4. The method of claim 3, wherein the animal of step 1) is a mouse, rat, pig or monkey. 4. The method of claim 3, wherein the animal of step 1) is a mouse generated from deposited fertilized eggs (Accession Number: KCTC 11247BP). 4. The method of claim 3, wherein the candidate material of step 1) is a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract or plasma. The method of claim 3, wherein the memory extinction ability test of step 2) is performed by periodically exposing the environment related to the conditioning after 1 hour to 60 hours after the conditioning reaction, and then measuring the freezing reaction. Characterized in that the method. 10. The method of claim 9, wherein the cycle is performed 20 to 30 times the conditioning reaction for 15 seconds to 50 seconds at intervals of 3 seconds to 180 seconds.

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