WO2019009586A1 - Depressive or epileptic animal model, establishment method therefor, and method for screening candidate drug for therapy of depressive disorder or epilepsy using same - Google Patents

Depressive or epileptic animal model, establishment method therefor, and method for screening candidate drug for therapy of depressive disorder or epilepsy using same Download PDF

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WO2019009586A1
WO2019009586A1 PCT/KR2018/007508 KR2018007508W WO2019009586A1 WO 2019009586 A1 WO2019009586 A1 WO 2019009586A1 KR 2018007508 W KR2018007508 W KR 2018007508W WO 2019009586 A1 WO2019009586 A1 WO 2019009586A1
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kcnk1
candidate drug
animal model
gene
epilepsy
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PCT/KR2018/007508
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French (fr)
Korean (ko)
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박재용
정현국
유강현
황은미
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고려대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to an animal model of depression or ephemeris using the CRISPR / Cas9 gene scissors technique and a method for preparing the same, and further to a method for screening a candidate drug for treating depression or brain damage using the animal model.
  • Depression is currently one of the most prevalent diseases, and the World Health Organization (WHO) is also one of the ten diseases that afflict humans in the 21st century. According to the National Health and Nutrition Examination Survey in 2001, about 3.2 million people, 8% of the total population, were found to suffer from depression. Sometimes people who feel depression were found to be 53.60%. In addition, the World Health Organization (WHO) has included depression in the world's 5th burden of disease by 2020.
  • depression The main symptom of depression is depression, helplessness, anxiety, loss of interest, appetite disorder, sleeping disorder, suicidal thoughts, and there is no sense of worth, guilt, concentration and memory.
  • Depression is also known to be accompanied by many complications such as obesity, cardiovascular disease, and degenerative nerve disease. Depression, helplessness, suicide, concentration and memory decline can lead to a loss of productivity as well as personal health, which can lead to enormous economic losses in society.
  • epilepsy or epilepsy is one of the chronic neurological disorders, and the rapid development of modern medicine has changed the concept of epilepsy (epilepsy) from a chronic illness that can not be cured in the past to a curable disease.
  • epileptic seizure The main symptom of epilepsy is the epileptic seizure and the epileptic seizure is caused by various factors such as focal brain lesion or systemic metabolic disorder, drug addiction, hypoxia, head trauma or extreme overwork and lack of sleep Is a central symptom caused by Epileptic seizures are defined as neurophysiologically sudden and disordered cerebral cortical cell dyssynchrony states, which can be easily understood clinically by seizure EEG findings.
  • Transgenic animals are animals that artificially insert an exogenous gene or remove a gene to express the intended genetic trait and can be useful in basic research to determine the role of a particular gene in living organisms . That is, it is possible to infer the physiological function mediated by the gene by inserting or deleting the gene whose function is suspected and observing how the effect appears.
  • transgenic animals can be used as models of human disease and are very useful in identifying the cause of a disease, the progress of a disease, and the effects or side effects of a potential therapeutic drug.
  • transgenic mice and zebrafish suffering from diseases such as dementia, cancer, heart disease, and genetic diseases have been developed (Korean Patent Registration No. 10-1750893).
  • the present inventors have made intensive efforts to develop an animal model of depression and / or epilepsy, and as a result, they have found that depressive symptoms occur uniformly in a transgenic animal model in which a specific gene is deleted.
  • Another object of the present invention is to provide a method for producing the animal model.
  • the present invention provides an animal model of depression or epilepsy, wherein the Kcnk1 (Potassium Channel Subfamily K) gene is a knock-out mouse.
  • the present invention also provides a method for producing a Kcnk1 gene-deficient mouse, wherein a CRISPR / Cas9 gene is removed from a chromosome on the basis of a part of exon 1 of Kcnk1 gene;
  • a method for producing an animal model of depression or epilepsy A method for producing an animal model of depression or epilepsy.
  • the present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
  • a method for screening candidate drugs for the prevention or treatment of depression or epilepsy A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
  • the present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
  • a method for screening candidate drugs for the prevention or treatment of depression or epilepsy A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
  • the present invention provides a method for preventing or treating depression or epilepsy in an animal model
  • the candidate drug may be a prophylactic or therapeutic agent for depression or epilepsy.
  • the candidate drug may not have a significant effect on the cognitive or learning ability of the control group.
  • mice lacking the Kcnk1 gene due to the CRISPR / Cas9 gene scissors according to the present invention had a part of the exon1 and the intron1 of the Kcnk1 gene removed, unlike the mouse lacking the Kcnk1 gene (exon2) produced by the conventional gene recombination technique, It is expected to be useful as an animal model of progression of depression and / or brain metastasis and pathological mechanism studies since depression and / or epilepsy are manifested and manifest symptoms of depression and / or brain metastasis.
  • mouse deficient in the Kcnk1 gene according to the present invention will be usefully used for the screening of candidate drugs for the prevention or treatment of depression and / or cerebral infarction.
  • FIG. 1A is a schematic diagram of genes of control and Kcnk1-deficient mice.
  • FIG. 1B shows the results of analysis of the Kcnk1 genotype in the control and Kcnk1-deficient mice.
  • FIG. 1C is a schematic diagram showing the sequence structure of mRNA expressed from the Kcnk1 gene in the control and Kcnk1-deficient mice.
  • FIG. 1d shows the results of RT-PCR analysis of mRNA expression from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice.
  • FIG. 1E shows the results of in situ hybridization analysis of mRNA expression from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice.
  • FIG. 1F shows the result of observing the protein expressed from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice by immunohistochemistry using a confocal microscope.
  • FIG. 2A shows the magnitude of the current mediated by the K + ions flowing to the cell membrane boundary due to the cell membrane potential change in astrocytes cultured from the brain of the control and Kcnk1-deficient mice.
  • FIG. 2B shows the values of extroversion current and inward current measured at cell membrane potential +50 mV and -150 mV, respectively, in FIG.
  • FIG. 2c is a graph directly showing the passive conductivity of astrocytes from the brain tissue sections of the control and Kcnk1-deficient mice.
  • FIG. 2d shows the magnitude of the current mediated by the K.sup. + Ions flowing to the cell membrane boundary according to the cell membrane potential change by analyzing the measured passive conductivity in FIG. 2c.
  • FIG. 2E shows extroversion current and inward current values measured at cell membrane potentials of +40 mV and -160 mV, respectively, in FIG. 2D.
  • Figures 3A-3C show the behavioral characteristics of the control and Kcnk1-deficient mice in an open field test.
  • Figure 3d shows the behavioral characteristics in the novel object recognition test of the control and Kcnk1-deficient mice.
  • Figure 3e shows behavioral characteristics in the elevated plus maze test of control and Kcnk1-deficient mice.
  • FIG. 3f shows the result of the sociability analysis in the 3-chamber social interaction test of the control and Kcnk1-deficient mice.
  • FIGS. 4A and 4B are results of analyzing seizure symptoms induced by injection of kainic acid (KA) in the control and Kcnk1-deficient mice.
  • KA kainic acid
  • Figures 4c and 4d are the results of analyzing seizure symptoms induced by injection of pentylenetetrazole (PTZ) in control and Kcnk1-deficient mice.
  • PTZ pentylenetetrazole
  • the present invention provides an animal model of depression or epilepsy, wherein the Kcnk1 (Potassium Channel Subfamily K) gene is a knock-out mouse.
  • the " Kcnk1 (Potassium Channel Subfamily K) " is also referred to as TWIK1 (tandem of P domains in a weak inward rectifier K channel 1) ).
  • TWIK1 tandem of P domains in a weak inward rectifier K channel 1 ).
  • the potassium (K) ion channel (potassium ion channel) exerts an important action in regulating the excitability of neuronal cells.
  • the ion base of the potassium ion channel is higher than the extracellular potassium ion concentration, After activation of the channel by depolarization, positively charged potassium ions flow out and the membrane potential is negatively charged (repolarization or hyperpolarization) and cell excitability is lowered.
  • Potassium ion channel anomalies can directly induce epilepsy, such as benign familial neonatal convulsions (BFNC).
  • BFNC benign familial neonatal convulsions
  • the above-mentioned " depression " is a disease in which negative emotions appear due to a change in the function of the brain that controls emotions.
  • the exact cause of the disease has not been clarified, and serotonin and melatonin as well as dopamine and norepinephrine Several hormones can affect depression.
  • the term " epileptic " refers to a group of chronic diseases in which some neurons generate excessive electricity in a short period of time due to synchronization with abnormal abnormalities and excitations of the brain and seizure occurs repeatedly. It is a serious neurological disorder with biological, mental, cognitive and social changes.
  • hippocampal sclerosis, gonadal hyperplasia (Hippocampal sclerosis, etc.) in the hippocampus of cerebral regions where pathological damage due to brain injury is evident due to excitotoxicity caused by hyperexcitability of abnormal neurons, gliosis, abnormal neurogenesis, cytoarchitectural abnormality of dentate granule cells, and aberrant syn- passic circuit.
  • pathologic events in the hippocampus may lead to chronic epileptic seizures and drug refractory intractable epilepsy that is not responsive to drug therapy as well as cognitive and memory disorders.
  • the present invention also provides a method for producing a Kcnk1 gene-deficient mouse, wherein a CRISPR / Cas9 gene is removed from a chromosome on the basis of a part of exon 1 of Kcnk1 gene;
  • a method for producing an animal model of depression or epilepsy A method for producing an animal model of depression or epilepsy.
  • CRISPR clustered, regular interspaced, short palindromic repeats
  • Cas9 CRISPR-associated 9
  • ZFNs Zinc-Finger Nucleases
  • TALENs Transcription Activator-like Effector Nucleases
  • the CRISPR / Cas9 system targets the target site in a specific gene of Cas9 by the induction of a single guide RNA (sgRNA) identical to the target site in a specific gene, thereby disrupting the function of the gene by causing gene insertion and loss Lt; / RTI >
  • sgRNA single guide RNA
  • the inventors of the present invention found that the preparation of an animal model of Kcnk1 deficient mice using the CRISPR / Cas9 gene scissors technique is superior to the production of KcnK1-deficient mice in which only a part of the protein produced from the target gene is removed by using a conventional gene recombination technique It was confirmed that most of the protein produced was removed (see Example 1).
  • the present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
  • a method for screening candidate drugs for the prevention or treatment of depression or epilepsy A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
  • the present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
  • a method for screening candidate drugs for the prevention or treatment of depression or epilepsy A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
  • the candidate drug may not significantly affect the recognition or learning ability of the control group.
  • the present invention also relates to a method for the treatment of depression or epilepsy, comprising administering a candidate drug to an animal model of depression or epilepsy;
  • a method for screening candidate drugs for the prevention or treatment of depression or epilepsy A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
  • the candidate drug may be, but not limited to, a drug for preventing or treating depression or epilepsy.
  • the " astrocyte " is one of the cells forming the glial glue that supports the nerve tissue. It is also called astrocytes, and is seen as a star due to the many protuberances and exists in the brain and spinal cord. Astrocytic cells help to biochemically support the inner cells of the blood-brain barrier (blood-brain barrier), and are attached to the walls of the blood vessels to supply nutrients to nerve cells. In addition, it plays various roles such as regulating the ion concentration of nerve cells, support of nerve cells, elimination of waste products, phagocytosis, etc. When the nerve tissue is damaged, it proliferates as glioma and fills the part, Or destroy it.
  • the K + ion-mediated current and the passive conductance of astrocytes in the brain tissue slices are decreased in astrocytes and brain slices derived from Kcnk1-deficient mice due to changes in cell membrane potential (See Example 2).
  • the animal model of Kcnk1-deficient mice was confirmed to exhibit the behavioral characteristics of depression (see Example 3) and the behavioral characteristics of the brain metastasis (see Example 4).
  • RNA designed to target a portion of 346 bp from the rear of exon 1 of the Kcnk1 gene to the front of intron 1, which was directly linked to the former, was removed by introducing CRISPR / Cas9, which cleaves the double stranded DNA.
  • CRISPR / Cas9 which cleaves the double stranded DNA.
  • Gentle embryos carrying the defective Kcnk1 gene were implanted into a surrogate mother to obtain a Kcnk1 gene-deficient mouse, and a Kcnk1 KO mouse in which Kcnk1 gene expression was completely deficient was obtained through intercrossing.
  • the genotypes of each mouse were confirmed by PCR using a set of primers recognizing the defective periphery.
  • the mRNA of Kcnk1 was not normally expressed in the brain tissue of the prepared Kcnk1-deficient mouse, which was confirmed by RT-PCR and in situ hybridization techniques and shown in FIGS. 1d and 1e.
  • Kcnk1 protein was observed under a microscope by brain immunohistochemistry to confirm expression of Kcnk1 protein in the Kcnk1-deficient mouse.
  • Kcnk1 KO mice and control mice were each anesthetized, perfused through the heart to remove the blood of brain tissue, and the tissues were fixed.
  • the brain tissue was sectioned to a thickness of about 40 ⁇ m by frozen embryo transfer method.
  • the brain tissue sections obtained above were stained with Kcnk1 and an antibody specifically recognizing GFAP known as astrocytic marker, and the expression of each protein was observed using a confocal fluorescence microscope.
  • Kcnk1 is an important ion channel responsible for passive conductivity by forming heterotopic complexes in astrocytes of the brain together with Kcnk2. Therefore, when the expression of Kcnk1 is deficient, it is expected that the electrophysiological characteristics such as the decrease of passive conductivity will be changed.
  • Kcnk1 - deficient mice We isolated and cultured astrocytes from Kcnk1 - deficient mice and measured the magnitude of the current mediated by K + ions flowing through cell membranes according to changes in cell membrane potential through electrophysiological techniques.
  • the electrode of the patch clamp device was approached to astrocytes and the current was measured by K + ion flowing through the cell membrane while keeping the fixed membrane potential at -60 mV and varying the membrane potential from -150 mV to +50 mV.
  • the voltage-current relationship is compared with the measured cell current by dividing the measured cell current by the cell capacitance of each astrocytic cell.
  • Kcnk1-deficient mice were kept at -60 mV from the astrocytes on the brain tissue slice, and the membrane potential was sequentially changed from -160 mV to +40 mV, and the K + ion-induced conductance (passive conductance) As a result of the measurement, it was confirmed that the passive conductivity was significantly reduced as compared with the control group as shown in FIG. 2C. As shown in FIG. 2d, the Kcnk1-deficient mice showed a much lower slope compared to the control group, and when compared with the current-voltage relationship (IV relationship), they showed +40 mV and -160 mV at the membrane potential was significantly reduced.
  • the present inventors have confirmed through a series of behavioral experiments that behavioral characteristics of depressive symptoms are shown in Kcnk1 deficient mice.
  • an open field test was conducted to analyze the behavior of animals in a certain open space.
  • One animal is placed in an open field surrounded by a square wall of 50 cm on one side, and the movement is tracked / photographed using a camcorder for 10 minutes.
  • the total distance and time of the experiment animals and the distance and time of movement at the edge or middle part of the open field were analyzed by video reading.
  • Kcnk1 KO mice and 9 control mice were used in the experiments, and the mean values between the two groups were compared and analyzed.
  • social interaction test was performed to analyze the sociality of animal models. More specifically, a mouse to be measured was placed in the middle of three consecutive rooms separated by a transparent wall, and another mouse object was placed in one of the two rooms. Then, the degree of interest of the measured subject to other subjects was measured by measuring the time toward the room with the other mouse object and the room facing the empty space.
  • Kcnk1 deficient mice had symptoms of depression compared to the control group.
  • KA Kainic acid
  • Kcnk1-deficient mice began to show the first seizure symptoms earlier than the control group.
  • FIG. 4B the number of animals showing seizures induced by the number of injections of ciphosphoric acid was more than twice that of the control group after the third injection of Kcnk1-deficient mice.
  • the seizure symptom induced by pentylenetradazole (PTZ) injection at 30 mg / kg was similar to that caused by KA.
  • the time until the appearance of the first seizure in the Kcnk1-deficient mouse was remarkably shorter than that in the control group, and in the seizure symptom analysis by the number of injections, the seizure symptoms were provoked by a small number of injections as shown in FIG. Respectively.
  • the mouse lacking the Kcnk1 gene by the CRISPR / Cas9 gene scissors according to the present invention can be used as an animal model of progression of depression and / or brain metastasis and pathological mechanism studies, and also can be used as an animal model of depression and / And can be usefully used for screening candidate drugs for prevention or treatment.

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Abstract

The present invention relates to a depressive or epileptic animal model and an establishment method therefor using CRISPR/Cas9 molecular scissors technology and further to a method for screening a candidate drug for therapy of depressive disorder or epilepsy using the animal model. Lacking most of the Kcnk1 gene, in contrast to the mice from which the Kcnk1 gene is partially deleted by classical genetic recombination techniques, Kcnk1 gene-knockout mice established using CRISPR/Cas9 molecular scissors according to the present invention show intrinsic symptoms of depressive disorder and/or epilepsy with the onset of depressive disorder and/or epilepsy therein and are expected to be useful as an animal model for research on the progression and pathological mechanism of depressive disorder and/or epilepsy. In addition, it is expected that the Kcnk1-knockout mice according to the present invention are effectively used for screening candidate drugs for prophylaxis or therapy of depressive disorder and/or epilepsy.

Description

우울증 또는 뇌전증 동물 모델과 그 제조방법 및 이를 이용한 우울증 또는 뇌전증 치료용 후보약물의 스크리닝 방법Animal model of depression or ephemeris, its manufacturing method, and screening method of candidate drug for treating depression or brain damage using the same
본 발명은 CRISPR/Cas9 유전자가위 기술을 사용한 우울증 또는 뇌전증 동물 모델과 그 제조방법에 관한 것이며, 더 나아가 상기 동물 모델을 이용하여 우울증 또는 뇌전증 치료용 후보약물을 스크리닝하는 방법에 관한 것이다.The present invention relates to an animal model of depression or ephemeris using the CRISPR / Cas9 gene scissors technique and a method for preparing the same, and further to a method for screening a candidate drug for treating depression or brain damage using the animal model.
우울증은 현재 유병률이 날로 증가하는 질환 중 하나로, 세계보건기구(WHO)도 21세기에 인류를 괴롭히는 10대 질병 중 하나로 우울증이 대두되고 있다. 우리나라 역시 2001년도 국민건강 영양조사에 의하면 전 국민의 8%인 약 320만 명이 항상 우울증에 시달린다고 밝혀졌다. 가끔 우울증을 느끼는 사람들로 53.60%나 되는 것으로 조사되었다. 또한 세계 보건기구(WHO)에서는 2020년 세계 5대 부담 질병에 우울증을 포함시켰다.Depression is currently one of the most prevalent diseases, and the World Health Organization (WHO) is also one of the ten diseases that afflict humans in the 21st century. According to the National Health and Nutrition Examination Survey in 2001, about 3.2 million people, 8% of the total population, were found to suffer from depression. Sometimes people who feel depression were found to be 53.60%. In addition, the World Health Organization (WHO) has included depression in the world's 5th burden of disease by 2020.
우울증의 주요 증상으로는 우울감, 무기력감, 불안, 흥미의 저하, 식욕장애, 수면장애, 자살 생각 등이 주요 증상으로 나타나며, 무가치감, 부적절한 죄책감, 집중력 및 기억력의 감퇴 등이 있다. 또한 우울증은 비만, 심혈관질환, 퇴행성신경질환 등 많은 합병증을 수반하는 것으로 알려져 있다. 우울증으로 인한 무기력감, 자살, 집중력 및 기억력의 감퇴는 개인의 건강뿐만 아니라 생산성 저하로 이어져 사회의 막대한 경제적 손실을 야기할 수 있다.The main symptom of depression is depression, helplessness, anxiety, loss of interest, appetite disorder, sleeping disorder, suicidal thoughts, and there is no sense of worth, guilt, concentration and memory. Depression is also known to be accompanied by many complications such as obesity, cardiovascular disease, and degenerative nerve disease. Depression, helplessness, suicide, concentration and memory decline can lead to a loss of productivity as well as personal health, which can lead to enormous economic losses in society.
한편, 간질 또는 뇌전증은 만성적인 신경 장애의 하나이며, 현대의학의 급격한 발전은 뇌전증(간질)의 개념을 과거의 치유될 수 없는 만성 질환으로부터 치유 가능한 질병으로 바꾸었다.On the other hand, epilepsy or epilepsy is one of the chronic neurological disorders, and the rapid development of modern medicine has changed the concept of epilepsy (epilepsy) from a chronic illness that can not be cured in the past to a curable disease.
뇌전증(간질)의 핵심을 이루는 증상은 간질성 발작(epileptic seizure)이며, 간질성 발작은 국소성 뇌병변이나 전신성 대사장애, 약물중독, 저산소증, 두부외상 또는 극심한 과로 및 수면부족 등 여러 가지 요인에 의해 발생되는 중추성 증상이다. 간질성 발작은 신경생리학적으로 갑작스럽고 무질서한 대뇌피질세포의 이상흥분상태라고 정의되는데, 이는 임상적으로 발작 뇌파 소견에 의해 쉽게 이해될 수 있다. 정상적인 기능을 하던 신경세포가 왜 갑작스러운 이상흥분상태로 전환하는가에 대해서는 아직 확실히 알려져 있지는 않지만 정상적 뇌기능을 위한 흥분과 억제의 균형 조절이 여러 가지 원인에 의해 깨지게 됨으로써 발생하는 것으로 이해되고 있고, 이는 실험적으로 흥분성 신경전달물질의 기능을 증가시키거나 또는 억제성 신경전달물질의 기능을 억압시킬 경우에 간질성 발작이 일어나게 되는 현상에 의해 뒷받침된다.The main symptom of epilepsy is the epileptic seizure and the epileptic seizure is caused by various factors such as focal brain lesion or systemic metabolic disorder, drug addiction, hypoxia, head trauma or extreme overwork and lack of sleep Is a central symptom caused by Epileptic seizures are defined as neurophysiologically sudden and disordered cerebral cortical cell dyssynchrony states, which can be easily understood clinically by seizure EEG findings. Although it is not yet clear why normal functioning neurons turn into a sudden abnormal excitatory state, it is understood that the regulation of the balance of excitement and inhibition for normal brain function is broken by various causes, This is supported by a phenomenon in which an epileptic seizure occurs when the function of the excitatory neurotransmitter is experimentally suppressed or the function of the inhibitory neurotransmitter is suppressed.
형질전환 동물(transgenic animals)은 인위적으로 외부의 유전자를 삽입하거나 또는 유전자를 제거하여 의도하는 유전형질을 발현시킨 동물을 말하며, 생명체에서의 특정 유전자의 역할을 파악하는 기초연구에 유용하게 쓰일 수 있다. 즉, 그 기능이 의심되는 유전자를 삽입 또는 결실시키고, 그 영향이 어떻게 나타나는지를 관찰하여 상기 유전자가 매개하는 생리적 기능을 유추할 수 있다. 뿐만 아니라, 형질전환 동물은 사람 질병의 모델로서 사용할 수 있고, 질병의 원인이나 질병의 진행과정 등을 파악하거나 잠재적 치료약품의 효과나 부작용을 알아보는데 매우 유용하다. 현재 치매, 암, 심장병, 유전병 등의 질병에 걸린 형질전환 마우스, 제브라피쉬 등이 개발되고 있다(국내등록특허 KR 10-1750893).Transgenic animals are animals that artificially insert an exogenous gene or remove a gene to express the intended genetic trait and can be useful in basic research to determine the role of a particular gene in living organisms . That is, it is possible to infer the physiological function mediated by the gene by inserting or deleting the gene whose function is suspected and observing how the effect appears. In addition, transgenic animals can be used as models of human disease and are very useful in identifying the cause of a disease, the progress of a disease, and the effects or side effects of a potential therapeutic drug. Currently, transgenic mice and zebrafish suffering from diseases such as dementia, cancer, heart disease, and genetic diseases have been developed (Korean Patent Registration No. 10-1750893).
이에, 본 발명자들은 신규한 우울증 및/또는 뇌전증 동물 모델을 개발하기 위하여 예의 노력한 결과, 특정 유전자가 결손된 형질전환 동물 모델에서 우울증 증상이 균일하게 발생하는 것을 확인하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive efforts to develop an animal model of depression and / or epilepsy, and as a result, they have found that depressive symptoms occur uniformly in a transgenic animal model in which a specific gene is deleted.
본 발명은 우울증 및/또는 뇌전증의 진행 및 병리학적 기전의 연구에 유용한 동물 모델을 제공하고자 하는 것을 목적으로 한다.It is an object of the present invention to provide an animal model useful for studying the progression of depression and / or brain metastasis and pathological mechanism.
본 발명은 또한 상기 동물 모델의 제조방법을 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a method for producing the animal model.
본 발명의 또 다른 목적은 상기 동물 모델을 이용하여 우울증 및/또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공하는 것이다.It is still another object of the present invention to provide a method for screening candidate drugs for the prevention or treatment of depression and / or ephemeris using the animal model.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems and other problems not mentioned can be clearly understood by those skilled in the art from the following description will be.
상기 목적을 달성하기 위하여, 본 발명은 Kcnk1(Potassium Channel Subfamily K) 유전자가 결손(knock-out)된 마우스인 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델을 제공한다.In order to achieve the above object, the present invention provides an animal model of depression or epilepsy, wherein the Kcnk1 (Potassium Channel Subfamily K) gene is a knock-out mouse.
또한, 본 발명은 Kcnk1 유전자 중 exon1 일부를 표적으로 하여 CRISPR/Cas9 유전자가위로 염색체 상에서 제거한 Kcnk1 유전자 결손 마우스를 얻는 단계;The present invention also provides a method for producing a Kcnk1 gene-deficient mouse, wherein a CRISPR / Cas9 gene is removed from a chromosome on the basis of a part of exon 1 of Kcnk1 gene;
상기 Kcnk1 유전자 결손 마우스 간에 교배를 통하여 2세대 마우스를 얻는 단계; 및Obtaining a second-generation mouse through cross-breeding among the Kcnk1 gene-deficient mice; And
상기 2세대 마우스로부터 Kcnk1 유전자가 발현되지 않는 마우스를 선별하는 단계;Selecting a mouse from which the Kcnk1 gene is not expressed from the second generation mouse;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델의 제조방법을 제공한다.A method for producing an animal model of depression or epilepsy.
또한, 본 발명은 상기 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;The present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
상기 후보약물 투여 후 상기 동물 모델의 발작 여부 및 생존율을 측정하는 단계; 및Measuring the seizure frequency and survival rate of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 발작을 완화시켜주거나 생존율을 향상시키는 후보약물을 선별하는 단계;Selecting a candidate drug that alleviates the seizure or improves the survival rate as compared with the control group in which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공한다.A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
또한, 본 발명은 상기 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;The present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
상기 후보약물 투여 후 상기 동물 모델의 행동학적 특성을 분석하는 단계; 및Analyzing the behavioral characteristics of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 우울증 증상을 완화시키는 행동학적 특성을 보이는 후보약물을 선별하는 단계;Selecting a candidate drug exhibiting a behavioral characteristic that alleviates the symptoms of depression as compared with a control group to which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공한다.A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
아울러, 본 발명은 상기 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;In addition, the present invention provides a method for preventing or treating depression or epilepsy in an animal model,
상기 후보약물 투여 후 상기 동물 모델의 별아교세포에서 수동전도도를 측정하는 단계; 및Measuring passive conductance in astrocytes of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 수동전도도를 증가시키는 후보약물을 선별하는 단계;Selecting a candidate drug that increases passive conductivity compared to a control to which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug is selected from the group consisting of: < RTI ID = 0.0 > (I) < / RTI >
본 발명의 일 구현예로, 상기 후보약물은 우울증 또는 뇌전증 예방 또는 치료제일 수 있다.In one embodiment of the invention, the candidate drug may be a prophylactic or therapeutic agent for depression or epilepsy.
본 발명의 일 구현예로, 상기 후보약물은 대조군의 인지 또는 학습 능력에 유의적인 영향을 미치지 않는 것일 수 있다.In one embodiment of the present invention, the candidate drug may not have a significant effect on the cognitive or learning ability of the control group.
본 발명에 따른 CRISPR/Cas9 유전자가위에 의하여 Kcnk1 유전자가 결손된 마우스는 전통적인 유전자재조합 기법으로 만들어진 Kcnk1 유전자 일부 (exon2)가 결손된 마우스와 달리 Kcnk1 유전자의 exon1의 일부와 intron1의 일부가 제거되었으며, 우울증 및/또는 뇌전증이 발병하여 우울증 및/또는 뇌전증 고유의 증상을 나타내므로 우울증 및/또는 뇌전증의 진행 경과와 병리학적 기전 연구의 동물 모델로 유용하게 이용될 것으로 기대된다.The mice lacking the Kcnk1 gene due to the CRISPR / Cas9 gene scissors according to the present invention had a part of the exon1 and the intron1 of the Kcnk1 gene removed, unlike the mouse lacking the Kcnk1 gene (exon2) produced by the conventional gene recombination technique, It is expected to be useful as an animal model of progression of depression and / or brain metastasis and pathological mechanism studies since depression and / or epilepsy are manifested and manifest symptoms of depression and / or brain metastasis.
또한, 본 발명에 따른 Kcnk1 유전자가 결손된 마우스는 우울증 및/또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝에 유용하게 이용될 것으로 기대된다.Further, it is expected that the mouse deficient in the Kcnk1 gene according to the present invention will be usefully used for the screening of candidate drugs for the prevention or treatment of depression and / or cerebral infarction.
도 1a는 대조군 및 Kcnk1 결손 마우스의 유전자의 모식도를 나타낸 것이다.FIG. 1A is a schematic diagram of genes of control and Kcnk1-deficient mice.
도 1b는 대조군 및 Kcnk1 결손 마우스에서 Kcnk1 유전자형을 분석한 결과이다.FIG. 1B shows the results of analysis of the Kcnk1 genotype in the control and Kcnk1-deficient mice.
도 1c는 대조군 및 Kcnk1 결손 마우스에서 Kcnk1 유전자로부터 발현되는 mRNA의 시퀀스 구조를 나타낸 모식도이다. FIG. 1C is a schematic diagram showing the sequence structure of mRNA expressed from the Kcnk1 gene in the control and Kcnk1-deficient mice.
도 1d는 대조군 및 Kcnk1 결손 마우스의 뇌조직에서 Kcnk1 유전자로부터의 mRNA 발현을 RT-PCR 기법을 통해 분석한 결과이다. FIG. 1d shows the results of RT-PCR analysis of mRNA expression from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice.
도 1e는 대조군 및 Kcnk1 결손 마우스의 뇌조직에서 Kcnk1 유전자로부터의 mRNA 발현을 in situ hybridization 기법을 통하여 분석한 결과이다. FIG. 1E shows the results of in situ hybridization analysis of mRNA expression from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice.
도 1f는 대조군 및 Kcnk1 결손 마우스의 뇌조직에서 Kcnk1 유전자로부터 발현되는 단백질을 면역염색법을 통해 공촛점현미경으로 관찰한 결과이다. FIG. 1F shows the result of observing the protein expressed from the Kcnk1 gene in the brain tissues of the control and Kcnk1-deficient mice by immunohistochemistry using a confocal microscope.
도 2a는 대조군 및 Kcnk1 결손 마우스의 뇌로부터 배양된 별아교세포에서 세포막전위 변화에 따른 세포막을 경계로 흐르는 K+이온에 의해 매개되는 전류의 크기를 나타낸 것이다.FIG. 2A shows the magnitude of the current mediated by the K + ions flowing to the cell membrane boundary due to the cell membrane potential change in astrocytes cultured from the brain of the control and Kcnk1-deficient mice.
도 2b는 도 2a의 세포막전위 +50 mV, -150 mV에서 각각 측정된 외향성 전류와 내향성 전류 값을 나타낸 것이다.FIG. 2B shows the values of extroversion current and inward current measured at cell membrane potential +50 mV and -150 mV, respectively, in FIG.
도 2c는 대조군 및 Kcnk1 결손 마우스의 뇌조직 절편으로부터 별아교세포의 수동적 전도도를 직접 측정한 값을 나타낸 것이다.FIG. 2c is a graph directly showing the passive conductivity of astrocytes from the brain tissue sections of the control and Kcnk1-deficient mice.
도 2d는 도 2c에서 측정된 수동전도도를 분석하여 세포막전위 변화에 따라 세포막을 경계로 흐르는 K+이온에 의해 매개되는 전류의 크기를 나타낸 것이다.FIG. 2d shows the magnitude of the current mediated by the K.sup. + Ions flowing to the cell membrane boundary according to the cell membrane potential change by analyzing the measured passive conductivity in FIG. 2c.
도 2e는 도 2d의 세포막전위 +40 mV, -160 mV에서 각각 측정된 외향성 전류와 내향성 전류 값을 나타낸 것이다.FIG. 2E shows extroversion current and inward current values measured at cell membrane potentials of +40 mV and -160 mV, respectively, in FIG. 2D.
도 3a 내지 도 3c는 대조군 및 Kcnk1 결손 마우스의 open field test에서 행동학적 특성을 나타낸 것이다.Figures 3A-3C show the behavioral characteristics of the control and Kcnk1-deficient mice in an open field test.
도 3d는 대조군 및 Kcnk1 결손 마우스의 novel object recognition test에서 행동학적 특성을 나타낸 것이다.Figure 3d shows the behavioral characteristics in the novel object recognition test of the control and Kcnk1-deficient mice.
도 3e는 대조군 및 Kcnk1 결손 마우스의 Elevated plus maze test에서 행동학적 특성을 나타낸 것이다.Figure 3e shows behavioral characteristics in the elevated plus maze test of control and Kcnk1-deficient mice.
도 3f는 대조군 및 Kcnk1 결손 마우스의 3 chamber social interaction test에서 사회성 분석 결과를 나타낸 것이다.FIG. 3f shows the result of the sociability analysis in the 3-chamber social interaction test of the control and Kcnk1-deficient mice.
도 4a 및 4b는 대조군 및 Kcnk1 결손 마우스에서 카인산 (kainic acid; KA) 주사를 통하여 유발되는 발작 (seizure) 증상을 분석하여 비교한 결과이다. FIGS. 4A and 4B are results of analyzing seizure symptoms induced by injection of kainic acid (KA) in the control and Kcnk1-deficient mice.
도 4c 및 4d는 대조군 및 Kcnk1 결손 마우스에서 펜틸렌테드라졸 (pentylenetetrazole; PTZ) 주사를 통하여 유발되는 발작 증상을 분석하여 비교한 결과이다. Figures 4c and 4d are the results of analyzing seizure symptoms induced by injection of pentylenetetrazole (PTZ) in control and Kcnk1-deficient mice.
본 발명은 Kcnk1(Potassium Channel Subfamily K) 유전자가 결손(knock-out)된 마우스인 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델을 제공한다.The present invention provides an animal model of depression or epilepsy, wherein the Kcnk1 (Potassium Channel Subfamily K) gene is a knock-out mouse.
본 발명에 있어서, 상기 “Kcnk1(Potassium Channel Subfamily K)”는 TWIK1(tandem of P domains in a weak inward rectifier K channel 1)이라고도 하며 potassium(K) 이온통로 중 two-pore domain K 이온통로군(K2P)에 속하는 이온통로이다.In the present invention, the " Kcnk1 (Potassium Channel Subfamily K) " is also referred to as TWIK1 (tandem of P domains in a weak inward rectifier K channel 1) ). ≪ / RTI >
이 때, 상기 potassium(K) 이온통로(칼륨 이온통로)는 신경세포의 흥분성을 조절하는 방면에서 중요한 작용을 발휘하는 바, 이의 이온기초는 세포 내 칼륨 이온 농도가 세포 외 칼륨 이온 농도보다 높고 막전위 탈분극에 의해 통로를 활성화시킨 후, 양전하를 띤 칼륨 이온은 밖으로 흐르고, 막전위는 이로 인해 음전하를 띠고(재분극 또는 과분극), 세포 흥분성은 하강된다. 칼륨 이온통로 이상은 양성 신생아 가족성 경련(BFNC, benign familial neonatal convulsion)과 같은 간질을 직접적으로 유발할 수 있다.At this time, the potassium (K) ion channel (potassium ion channel) exerts an important action in regulating the excitability of neuronal cells. The ion base of the potassium ion channel is higher than the extracellular potassium ion concentration, After activation of the channel by depolarization, positively charged potassium ions flow out and the membrane potential is negatively charged (repolarization or hyperpolarization) and cell excitability is lowered. Potassium ion channel anomalies can directly induce epilepsy, such as benign familial neonatal convulsions (BFNC).
본 발명에 있어서, 상기 “우울증”은 감정을 조절하는 뇌의 기능에 변화가 생겨 부정적인 감정이 나타나는 병으로, 정확한 발병 원인은 밝혀지지 않았으며, 세로토닌 및 멜라토닌을 비롯하여 도파민, 노르에피네프린 등 신경과 관련된 여러 가지 호르몬이 우울증에 영향을 미칠 수 있다.In the present invention, the above-mentioned " depression " is a disease in which negative emotions appear due to a change in the function of the brain that controls emotions. The exact cause of the disease has not been clarified, and serotonin and melatonin as well as dopamine and norepinephrine Several hormones can affect depression.
본 발명에 있어서, 상기 “뇌전증”은 뇌의 비정상적인 과흥분과 과동기화로 인해 신경세포 중 일부가 짧은 시간에 과도한 전기를 발생시켜 반복적으로 발작(seizure)이 발생하는 만성화된 질환군으로, 신경생물학적, 정신적, 인지적, 사회적 변화를 수반하는 심각한 신경 질환이다. 또한, 신경세포의 비정상적 과다흥분성(hyperexcitability)에 의한 흥분독성(excitotoxicity)이 유발되어 뇌전증에 의한 병리학적 손상이 잘 나타나는 대뇌 부위 중 해마(hippocampus) 내에서 해마경화증(hippocampal sclerosis), 교질화(gliosis), 비정상적 신경발생(abnormal neurogenesis)과 치아이랑 과립세포 비정상적 세포구축(cytoarchitectural abnormality of dentate granule cells) 및 이상시냅스회로 형성(aberrant synpatic circuit)등이 나타난다. 위와 같은 해마 내 병리학적 현상들이 진행됨으로서 만성 뇌전증 발작(chronic epileptic seizure)이 발생되고 인지 및 기억 장애뿐만 아니라 약물 치료에 반응하지 않는 약제불응성 난치성 뇌전증이 발생된다.In the present invention, the term " epileptic " refers to a group of chronic diseases in which some neurons generate excessive electricity in a short period of time due to synchronization with abnormal abnormalities and excitations of the brain and seizure occurs repeatedly. It is a serious neurological disorder with biological, mental, cognitive and social changes. In addition, hippocampal sclerosis, gonadal hyperplasia (Hippocampal sclerosis, etc.) in the hippocampus of cerebral regions, where pathological damage due to brain injury is evident due to excitotoxicity caused by hyperexcitability of abnormal neurons, gliosis, abnormal neurogenesis, cytoarchitectural abnormality of dentate granule cells, and aberrant syn- passic circuit. These pathologic events in the hippocampus may lead to chronic epileptic seizures and drug refractory intractable epilepsy that is not responsive to drug therapy as well as cognitive and memory disorders.
또한, 본 발명은 Kcnk1 유전자 중 exon1 일부를 표적으로 하여 CRISPR/Cas9 유전자가위로 염색체 상에서 제거한 Kcnk1 유전자 결손 마우스를 얻는 단계;The present invention also provides a method for producing a Kcnk1 gene-deficient mouse, wherein a CRISPR / Cas9 gene is removed from a chromosome on the basis of a part of exon 1 of Kcnk1 gene;
상기 Kcnk1 유전자 결손 마우스 간에 교배를 통하여 2세대 마우스를 얻는 단계; 및Obtaining a second-generation mouse through cross-breeding among the Kcnk1 gene-deficient mice; And
상기 2세대 마우스로부터 Kcnk1 유전자가 발현되지 않는 마우스를 선별하는 단계;Selecting a mouse from which the Kcnk1 gene is not expressed from the second generation mouse;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델의 제조방법을 제공한다.A method for producing an animal model of depression or epilepsy.
본 발명에 있어서, 상기 “CRISPR(clustered, regular interspaced, short palindromic repeats)/Cas9 (CRISPR-associated 9)” 시스템은 1987년에 외부 플라스미드와 바이러스의 분해와 관련된 ‘면역 시스템’으로 처음 발견되었으며, 그 이후로 상기 시스템은 여러 응용이 가능하고 매우 효율적인 유전체 편집 기술로 널리 사용되어져 왔다. 이는 흔히 유전자 가위라 불리는 endonuclease splicing 기술로서 1세대인 Zinc-Finger Nucleases (ZFNs)와, 2세대인 Transcription Activator-like Effector Nucleases (TALENs)보다 효율적이고 손쉽게 유전자를 조작할 수 있는 이점이 있다. 이러한 CRISPR/Cas9 시스템은 특정 유전자 내에 target site와 동일한 단일 가이드 RNA (single guide RNA; sgRNA)의 유도에 의해 Cas9의 특정 유전자 내 target site를 공격하여 유전자 삽입과 손실을 일으켜 유전자의 기능을 무력화하는 원리로 작동한다. 이와 같이 만약 염색체 내 특정 부분에 위치한 유전자에 인위적으로 삽입, 손실, 변형 등을 유도할 수 있다면 유전학 및 분자 생물학 연구뿐만 아니라 특정 유전자와 관련된 질병 연구에도 널리 활용될 수 있다.In the present invention, the "CRISPR (clustered, regular interspaced, short palindromic repeats) / Cas9 (CRISPR-associated 9)" system was first found in 1987 as an "immune system" related to the degradation of outer plasmids and viruses, Since then, the system has been widely used as a highly efficient dielectric editing technique that is capable of many applications. This is an endonuclease splicing technique commonly called gene scissors, which is more efficient and easier to manipulate genes than the first-generation Zinc-Finger Nucleases (ZFNs) and the second-generation Transcription Activator-like Effector Nucleases (TALENs). The CRISPR / Cas9 system targets the target site in a specific gene of Cas9 by the induction of a single guide RNA (sgRNA) identical to the target site in a specific gene, thereby disrupting the function of the gene by causing gene insertion and loss Lt; / RTI > Thus, if artificial insertion, loss, and transformation of a gene located in a specific part of a chromosome can be induced, it can be widely used for genetic and molecular biology research as well as disease research related to a specific gene.
본 발명자들은 CRISPR/Cas9 유전자가위 기술을 활용하여 Kcnk1 결손 마우스 동물 모델을 제조하는 것이 전통적인 유전자재조합 기법을 이용하여 표적 유전자로부터 만들어지는 단백질의 일부분만을 제거한 KcnK1 결손 마우스를 제조하는 것과 비교하여 표적 유전자로부터 만들어지는 단백질의 대부분을 제거함을 확인하였다(실시예 1 참조). The inventors of the present invention found that the preparation of an animal model of Kcnk1 deficient mice using the CRISPR / Cas9 gene scissors technique is superior to the production of KcnK1-deficient mice in which only a part of the protein produced from the target gene is removed by using a conventional gene recombination technique It was confirmed that most of the protein produced was removed (see Example 1).
또한, 본 발명은 상기 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;The present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
상기 후보약물 투여 후 상기 동물 모델의 발작 여부 및 생존율을 측정하는 단계; 및Measuring the seizure frequency and survival rate of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 발작을 완화시켜주거나 생존율을 향상시키는 후보약물을 선별하는 단계;Selecting a candidate drug that alleviates the seizure or improves the survival rate as compared with the control group in which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공한다.A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
또한, 본 발명은 상기 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;The present invention also provides a method for preventing or treating depression or epilepsy, comprising administering a candidate drug to the depressive or epileptic animal model;
상기 후보약물 투여 후 상기 동물 모델의 행동학적 특성을 분석하는 단계; 및Analyzing the behavioral characteristics of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 우울증 증상을 완화시키는 행동학적 특성을 보이는 후보약물을 선별하는 단계;Selecting a candidate drug exhibiting a behavioral characteristic that alleviates the symptoms of depression as compared with a control group to which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공한다.A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
이 때, 상기 후보약물은 대조군의 인지 또는 학습 능력에 유의적인 영향을 미치지 않는 것일 수 있다.At this time, the candidate drug may not significantly affect the recognition or learning ability of the control group.
또한, 본 발명은 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;The present invention also relates to a method for the treatment of depression or epilepsy, comprising administering a candidate drug to an animal model of depression or epilepsy;
상기 후보약물 투여 후 상기 동물 모델의 별아교세포에서 수동전도도를 측정하는 단계; 및Measuring passive conductance in astrocytes of the animal model after administration of the candidate drug; And
상기 후보약물을 투여하지 않은 대조군과 비교하여 수동전도도를 증가시키는 후보약물을 선별하는 단계;Selecting a candidate drug that increases passive conductivity compared to a control to which the candidate drug is not administered;
를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법을 제공한다.A method for screening candidate drugs for the prevention or treatment of depression or epilepsy.
본 발명에 있어서, 상기 후보약물은 우울증 또는 뇌전증 예방 또는 치료제일 수 있으나, 이에 제한되지 않는다.In the present invention, the candidate drug may be, but not limited to, a drug for preventing or treating depression or epilepsy.
본 발명에 있어서, 상기 “별아교세포(astrocyte)”는 신경 조직을 지지하는 신경 아교를 이루는 세포 중 하나로 성상교세포라고도 하며, 뻗어있는 많은 돌기 때문에 별처럼 보이고 뇌와 척수에 존재한다. 별아교세포는 혈뇌장벽(혈액-뇌 장벽)의 안쪽 세포들을 생화학적으로 도와주는 역할을 하며 혈관벽에 돌기가 붙어있어 신경세포에 영양분을 공급하는 역할을 한다. 또한, 신경세포의 이온농도 조절, 신경세포의지지, 노폐물의 제거, 식세포작용 등의 다양한 역할을 수행하며, 신경조직이 손상되면 신경교종이라는 돌기로 증식하여 그 부분을 채우기도 하고 손상된 조직을 복구하거나 파괴하는 역할을 한다.In the present invention, the " astrocyte " is one of the cells forming the glial glue that supports the nerve tissue. It is also called astrocytes, and is seen as a star due to the many protuberances and exists in the brain and spinal cord. Astrocytic cells help to biochemically support the inner cells of the blood-brain barrier (blood-brain barrier), and are attached to the walls of the blood vessels to supply nutrients to nerve cells. In addition, it plays various roles such as regulating the ion concentration of nerve cells, support of nerve cells, elimination of waste products, phagocytosis, etc. When the nerve tissue is damaged, it proliferates as glioma and fills the part, Or destroy it.
본 발명의 일 실시예에서는 Kcnk1 결손 마우스로부터 유래된 별아교세포와 뇌절편에서 세포막전위 변화에 따라 세포막을 경계로 흐르는 K+이온에 의해 매개되는 전류와 뇌조직 절편에서 별아교세포의 수동전도도가 감소되어 있음을 확인하였다(실시예 2 참조). In one embodiment of the present invention, the K + ion-mediated current and the passive conductance of astrocytes in the brain tissue slices are decreased in astrocytes and brain slices derived from Kcnk1-deficient mice due to changes in cell membrane potential (See Example 2).
본 발명의 다른 실시예에서는 Kcnk1 결손 마우스 동물 모델이 우울증의 행동학적 특성(실시예 3 참조) 및 뇌전증의 행동학적 특성(실시예4 참조)을 보임을 확인하였다. In another embodiment of the present invention, the animal model of Kcnk1-deficient mice was confirmed to exhibit the behavioral characteristics of depression (see Example 3) and the behavioral characteristics of the brain metastasis (see Example 4).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
실시예 1. CRISPR/Cas9 유전자가위 기술을 활용한 Kcnk1 결손 마우스의 제조Example 1. Preparation of Kcnk1-deficient mice utilizing the CRISPR / Cas9 gene scissors technique
Kcnk1 유전자의 exon1의 뒷부분으로부터 바로 연결되어 있는 intron1의 앞부분에 이르는 346 bp에 해당하는 부분을 표적으로 하도록 설계된 가이드 RNA (sgRNA)와 함께 이중나선의 DNA를 절단하는 CRISPR/Cas9을 도입하여 제거하였다. 이렇게 결손된 Kcnk1 유전자를 지니는 배아세포를 대리모에 착상시켜 Kcnk1 유전자 결손 마우스를 얻고, 이들 간의 교배를 통하여 Kcnk1 유전자 발현이 완전히 결핍된 Kcnk1 KO 마우스를 얻었다. 각 마우스의 유전자형은 결손된 주변을 인식하는 한 조의 프라이머를 이용하여 PCR 기법을 통하여 확인하였다. (SgRNA) designed to target a portion of 346 bp from the rear of exon 1 of the Kcnk1 gene to the front of intron 1, which was directly linked to the former, was removed by introducing CRISPR / Cas9, which cleaves the double stranded DNA. Gentle embryos carrying the defective Kcnk1 gene were implanted into a surrogate mother to obtain a Kcnk1 gene-deficient mouse, and a Kcnk1 KO mouse in which Kcnk1 gene expression was completely deficient was obtained through intercrossing. The genotypes of each mouse were confirmed by PCR using a set of primers recognizing the defective periphery.
그 결과, 도 1a에 나타난 바와 같이, 염색체 상에서 Kcnk1 유전자 중 exon1과 intron1의 일부분이 제거된 Kcnk1 결손 마우스를 얻었다.As a result, as shown in FIG. 1A, Kcnk1-deficient mice in which exon 1 and intron 1 were partially removed from the Kcnk1 gene on the chromosome were obtained.
상기 Kcnk1 결손 마우스의 게놈(genome) 상에 표적으로 한 Kcnt1 유전자의 부분이 완전히 제거되었음을 확인하기 위해 PCR 기법을 이용하여 표현형을 확인하였다.In order to confirm that the target Kcnt1 gene was completely removed on the genome of the Kcnk1-deficient mouse, the phenotype was confirmed by PCR.
그 결과, 도 1b에 나타난 바와 같이, 상기 제조한 Kcnk1 결손 마우스의 게놈 상에 표적으로 한 Kcnt1 유전자의 부분이 완전히 제거되었음을 확인하였다.As a result, it was confirmed that the target Kcnt1 gene was completely removed from the genome of the Kcnk1-deficient mouse as shown in FIG. 1B.
따라서, 도 1c에서 나타난 바와 같이, 제조된 Kcnk1 결손 마우스의 뇌조직에서 Kcnk1의 mRNA는 정상적으로 발현되지 못하고, 이를 RT-PCR 기법과 in situ hybridization 기법을 통해 확인하여 도 1d 및 1e에 나타내었다.Thus, as shown in FIG. 1C, the mRNA of Kcnk1 was not normally expressed in the brain tissue of the prepared Kcnk1-deficient mouse, which was confirmed by RT-PCR and in situ hybridization techniques and shown in FIGS. 1d and 1e.
또한, 상기 Kcnk1 결손 마우스에서 Kcnk1 단백질이 발현되는지 확인하기 위하여 뇌조직 면역염색법을 통하여 Kcnk1 단백질을 현미경으로 관찰하였다. Kcnk1 KO 마우스와 대조군 마우스를 각각 마취 후 심장을 통한 관류 (perfusion)을 통해 뇌조직의 혈액을 제거하고 조직을 고정시킨 후, 동결박편법으로 뇌조직을 약 40 μm 두께로 절편화 하였다. 상기로부터 얻은 뇌조직 절편을 Kcnk1과 별아교세포 표지자로 알려진 GFAP를 특이적으로 인식하는 항체를 사용하여 염색한 후 공촛점형광현미경을 이용하여 각 단백질의 발현여부를 관찰하였다. Further, Kcnk1 protein was observed under a microscope by brain immunohistochemistry to confirm expression of Kcnk1 protein in the Kcnk1-deficient mouse. Kcnk1 KO mice and control mice were each anesthetized, perfused through the heart to remove the blood of brain tissue, and the tissues were fixed. The brain tissue was sectioned to a thickness of about 40 μm by frozen embryo transfer method. The brain tissue sections obtained above were stained with Kcnk1 and an antibody specifically recognizing GFAP known as astrocytic marker, and the expression of each protein was observed using a confocal fluorescence microscope.
그 결과, 도 1f에 나타난 바와 같이, 상기 Kcnk1 결손 마우스에서 Kcnk1 단백질이 발현되지 않음을 확인하였다.As a result, as shown in Fig. 1F, it was confirmed that the Kcnk1 protein was not expressed in the Kcnk1-deficient mouse.
실시예 2. Kcnk1 결손 마우스의 전기생리학적 특성 확인Example 2. Confirmation of electrophysiological characteristics of Kcnk1 deficient mice
Kcnk1은 Kcnk2와 함께 뇌의 별아교세포에서 이형결합체를 형성함으로써, 수동전도도를 담당하는 중요한 이온통로임이 잘 알려져 있다. 따라서 Kcnk1의 발현이 결핍될 경우 수동전도도 감소 등의 전기생리학적 특성의 변화가 일어날 것으로 예상할 수 있다. 이에, Kcnk1 결손 마우스로부터 별아교세포를 분리, 배양한 후 전기생리학적 기법을 통해 세포막전위 변화에 따라 세포막을 경계로 흐르는 K+이온에 의해 매개되는 전류의 크기를 측정하였다. 패치클램프 장비의 전극을 별아교세포에 접근시킨 후 전세포 방식으로 고정막전위를 -60 mV로 잡고 막전위를 -150 mV ~ +50 mV 까지 변화시키면서 세포막을 통해 흐르는 K+ 이온에 의한 전류를 측정하였다. 이렇게 측정된 전류의 크기를 각 별아교세포가 가지고 있는 전기용량 (cell capacitance)으로 나눈 값을 각 막전위에 대응시켜 전압-전류관계를 비교하여 도 2a에 나타내었다. It is well known that Kcnk1 is an important ion channel responsible for passive conductivity by forming heterotopic complexes in astrocytes of the brain together with Kcnk2. Therefore, when the expression of Kcnk1 is deficient, it is expected that the electrophysiological characteristics such as the decrease of passive conductivity will be changed. We isolated and cultured astrocytes from Kcnk1 - deficient mice and measured the magnitude of the current mediated by K + ions flowing through cell membranes according to changes in cell membrane potential through electrophysiological techniques. The electrode of the patch clamp device was approached to astrocytes and the current was measured by K + ion flowing through the cell membrane while keeping the fixed membrane potential at -60 mV and varying the membrane potential from -150 mV to +50 mV. The voltage-current relationship is compared with the measured cell current by dividing the measured cell current by the cell capacitance of each astrocytic cell.
그 결과, 대조군 마우스로 부터 유래된 별아교세포에 비하여 Kcnk1 KO 마우스로부터 배양된 별아교세포에서는 각 막전위에서 외향성 혹은 내향성 전류의 크기가 현저히 감소됨을 보였고, +50 mV와 150 mV의 막전위에서 외향성 전류와 내향성 전류를 측정한 결과, 도 2b에 나타난 바와 같이, 외향성 전류와 내향성 전류가 각각 현격히 감소되어 있음을 확인하였다. 이는 Kcnk1 이온통로의 발현결핍으로 인한 별아교세포의 전기생리학적 기능이 저해되었음을 의미한다. As a result, extracellular or introductory currents at each membrane potential were markedly reduced in astrocytes cultured from Kcnk1 KO mice compared with astrocytes derived from control mice. The extracellular currents and introductory currents at +50 mV and 150 mV As a result of measuring the current, it was confirmed that the extroversion current and the inward current were significantly reduced as shown in FIG. 2B. This means that the electrophysiological function of astrocytes due to the deficiency of Kcnk1 ion channel expression is impaired.
또한, Kcnk1 결손 마우스의 뇌조직 절편 상의 별아교세포로부터 고정막전위를 -60 mV로 유지한 채 막전위를 -160 mV ~ +40 mV로 순차적으로 변화시키면서 K+이온에 의한 전도도 (수동전도도; passive conductance)를 측정한 결과, 도 2c에 나타난 바와 같이 수동전도도가 대조군과 비교하여 현저하게 감소되어 있음을 확인하였다. 이를 다시 분석하여 전류-전압 관계(I-V relationship)로 비교하였을 때, 도 2d에 나타난 바와 같이 Kcnk1 결손 마우스의 경우 대조군에 비해 훨씬 낮은 기울기를 보였으며, 도 2e에 나타난 바와 같이 +40 mV와 -160 mV의 막전위에서의 K+ 전도도 값이 크게 감소하였음을 확인하였다.In addition, Kcnk1-deficient mice were kept at -60 mV from the astrocytes on the brain tissue slice, and the membrane potential was sequentially changed from -160 mV to +40 mV, and the K + ion-induced conductance (passive conductance) As a result of the measurement, it was confirmed that the passive conductivity was significantly reduced as compared with the control group as shown in FIG. 2C. As shown in FIG. 2d, the Kcnk1-deficient mice showed a much lower slope compared to the control group, and when compared with the current-voltage relationship (IV relationship), they showed +40 mV and -160 mV at the membrane potential was significantly reduced.
실시예 3. Kcnk1 결손 마우스의 우울증 관련 행동학적 특성 확인Example 3: Depression-related behavioral characteristics of Kcnk1 deficient mice
본 발명자들은 일련의 행동실험을 통하여 Kcnk1 결손 마우스에 우울증 증상의 행동학적 특성이 보임을 확인하였다. The present inventors have confirmed through a series of behavioral experiments that behavioral characteristics of depressive symptoms are shown in Kcnk1 deficient mice.
우선, 열린 일정한 공간에 동물을 위치시키고 일정시간 동안 동물의 활동양상을 분석하기 위해 open field test를 실시하였다. 한 변의 길이가 50 cm인 정사각형의 벽으로 둘러싸인 open field에 실험동물을 한 마리 씩 넣고, 이동을 캠코더를 사용하여 10 분 동안 추적/촬영한다. 비디오 판독을 통하여 실험동물의 총 이동거리 및 시간, open field에서 가장자리 혹은 가운데 부분에서의 이동거리 및 시간을 분석하였다. Kcnk1 KO 마우스와 대조군 마우스는 각각 11 마리와 9 마리를 실험에 사용하였고, 두 군 간의 평균값을 서로 비교 분석하였다.First, an open field test was conducted to analyze the behavior of animals in a certain open space. One animal is placed in an open field surrounded by a square wall of 50 cm on one side, and the movement is tracked / photographed using a camcorder for 10 minutes. The total distance and time of the experiment animals and the distance and time of movement at the edge or middle part of the open field were analyzed by video reading. Kcnk1 KO mice and 9 control mice were used in the experiments, and the mean values between the two groups were compared and analyzed.
그 결과, 도 3a 내지 도 3c에 나타난 바와 같이, open field test에서 Kcnk1 결손 마우스는 대조군에 비하여, 공간의 가운데 부분에서의 이동거리와 머문 시간이 현저히 낮음을 확인하였다. 이는, 우울증 및 불안증 증상과 유사한 증상이다.As a result, as shown in Figs. 3A to 3C, in the open field test, it was confirmed that the movement distance and the time spent in the center of the space were significantly lower in the Kcnk1-deficient mouse than in the control group. This is a symptom similar to depression and anxiety symptoms.
또한, 새로운 물체에 대한 실험동물의 호기심에 의한 행동 양식을 분석하는 novel object recognition test를 실시하였다. 투명한 벽으로 구분된 3 개의 연속된 방으로 이루어진 실험장의 가운데 방에 실험동물을 위치시키고, 양 쪽 2 개의 방 중에 한 쪽에만 실험동물에게 노출시킨 적 없는 생소한 물체(novel object)를 넣는다. 실험동물의 행동을 1 시간 동안 캠코더로 촬영하고, 양 쪽 방을 향해 머무는 시간을 각각 측정하여 새로운 물체에 관심을 보이는 정도를 분석하였다.In addition, a novel object recognition test was conducted to analyze the curiosity behavior of experimental animals on new objects. Place the experimental animals in the middle room of a three-chambered room separated by a transparent wall and place a novel object that has not been exposed to the laboratory animal in only one of the two rooms. The behavior of the experimental animals was photographed with a camcorder for 1 hour and the time spent staying in both rooms was measured to analyze the degree of interest in new objects.
그 결과, 도 3d에 나타난 바와 같이, Kcnk1 결손 마우스가 대조군과 비교하여 인지 및 학습 능력에 차이는 없으나, 새로운 물체에 관심을 보이는 시간이 현저히 적음을 관찰함으로써 새로운 물체에 대한 호기심이 감소되어 있음을 확인하였다.As a result, as shown in FIG. 3D, there was no difference in cognition and learning ability between the Kcnk1-deficient mouse and the control group, but the curiosity of the new object was reduced by observing that the time of interest in the new object was remarkably small Respectively.
한편, elevated plus maze test를 이용하여 Kcnk1 KO 마우스의 우울증/불안증의 행동학적 특성을 분석하였다. 실험동물이 임의로 내려올 수 없도록 지면으로 부터 50 cm 상공에 폭 10 cm, 길이 50 cm의 4개의 통로를 십자로 교차시켜 설치하고, 이중 마주 보는 2개의 통로에는 높이 40 cm의 벽을 설치하여 패쇄 통로로, 나머지 마주 보는 2개의 통로는 개방통로로 만들었다. 이런 elevated plus maze 위에 실험동물을 위치시키고, 대상 실험동물의 움직임을 10분 동안 캠코더로 추적 및 촬영하였다. 촬영된 동영상을 분석하여, 실험동물이 어느 통로에서 더 많은 시간을 머무는지를 측정하고 분석하였다. 그 결과, 도 3e에 나타난 바와 같이, Kcnk1 결손 마우스는 대조군과 비교하여 폐쇄된 통로에서 더 오랜 시간을 보내나, 반면 열린 공간에서는 더 짧은 시간 동안 머물고 있음을 확인하였다. 상기 결과를 통하여 Kcnk1 KO 마우스는 우울증 및 불안증 증상을 보임을 확인하였다. On the other hand, behavioral characteristics of depression / anxiety in Kcnk1 KO mice were analyzed using elevated plus maze test. In order to prevent the animals from falling down freely, four passageways 10 cm wide and 50 cm long are crossed over 50 cm above the ground, and 40 cm height walls are installed on the two opposite passageways. , And the other two opposite corridors were made open. The animals were placed on the elevated plus maze, and the movement of the animals was tracked and photographed with a camcorder for 10 minutes. The captured images were analyzed to determine and measure the passage times of the experimental animals. As a result, as shown in FIG. 3E, it was confirmed that the Kcnk1-deficient mice spent longer time in the closed passages, while staying in the open space for a shorter time as compared with the control group. These results indicate that Kcnk1 KO mice show depression and anxiety symptoms.
또한, 동물 모델의 사회성을 분석하기 위해 social interaction test를 실시하였다. 보다 구체적으로, 투명한 벽을 통해 분리된 연속된 3 개의 방 중 가운데 방에 측정 대상 마우스를 위치시키고, 양쪽 방 중 한 쪽 방에 다른 마우스 개체를 넣어주었다. 그 후 측정 대상 마우스가 다른 개체에 어느 정도 관심을 보이는지를 다른 마우스 개체가 있는 방 쪽과 비어있는 방쪽을 향해 있는 시간을 측정하여 분석하였다.In addition, social interaction test was performed to analyze the sociality of animal models. More specifically, a mouse to be measured was placed in the middle of three consecutive rooms separated by a transparent wall, and another mouse object was placed in one of the two rooms. Then, the degree of interest of the measured subject to other subjects was measured by measuring the time toward the room with the other mouse object and the room facing the empty space.
그 결과, 도 3f에 나타난 바와 같이, Kcnk1 결손 마우스는 대조군과 비교하여 낮은 사회성을 보였다.As a result, as shown in Fig. 3F, Kcnk1-deficient mice showed low sociability compared with the control group.
상기의 Kcnk1 결손 마우스의 행동학적 특성 결과를 종합해본 결과, Kcnk1 결손 마우스는 대조군에 비하여 우울증의 증상을 가지고 있음을 확인하였다. As a result of collecting the behavioral characteristics of the Kcnk1-deficient mouse, Kcnk1 deficient mice had symptoms of depression compared to the control group.
실시예 4. Kcnk1 결손 마우스의 뇌전증 관련 증상의 행동학적 특성 확인Example 4. Identification of Behavioral Characteristics of Epigastric Symptoms in Kcnk1-Deficient Mice
Kcnk1 결손에 의한 뇌전증 증상에 대한 영향을 평가하기 위하여 약물에 의해 유도된 발작 증상을 관찰 및 측정하여 분석하였다. 카인산 (KA)을 11 mg/kg 용량으로 대조군 및 Kcnk1 결손 마우스에 매 30 분마다 복강 주사 (intraperitoneal injection)하고 연속적으로 발작증상을 관찰하며 발작증상 정도를 측정하였다. In order to evaluate the effect of Kcnk1 deficiency on epigastric symptoms, drug - induced seizure symptoms were observed and measured. Kainic acid (KA) was intraperitoneally injected into the control and Kcnk1-deficient mice at a dose of 11 mg / kg every 30 minutes, and the severity of seizure symptoms was measured continuously.
그 결과, 도 4a에 나타난 바와 같이 Kcnk1 결손 마우스는 대조군에 비하여 첫 번째 발작 증상을 더 일찍 보이기 시작하였다. 또한 카인산 주사 횟수 별 발작 유발 동물의 수를 비교해 봤을 때, 도 4b에 나타난 바와 같이 Kcnk1 결손 마우스의 경우 3 번째 주사 후 대조군에 비해 2 배가 넘는 수의 동물에서 발작 증상이 나타남을 확인하였다. As a result, as shown in FIG. 4A, Kcnk1-deficient mice began to show the first seizure symptoms earlier than the control group. As shown in FIG. 4B, the number of animals showing seizures induced by the number of injections of ciphosphoric acid was more than twice that of the control group after the third injection of Kcnk1-deficient mice.
아울러, 30 mg/kg의 펜틸렌테드라졸 (PTZ) 주사를 통해 유발되는 발작 증상 측정에서도 KA에 의한 증상과 유사한 경향을 보였다. 도 4c에 나타난 바와 같이 Kcnk1 결손 마우스에서 첫 번째 발작이 나타나기까지의 시간이 대조군에 비해 현저히 짧았고, 주사 횟수 별 발작 증상 분석에서도 도 4d에 나타난 바와 같이 적은 횟수의 주사로도 발작 증상이 유발됨을 확인하였다. 이를 통해 Kcnk1 결손 마우스가 대조군에 비해 쉽게 뇌전증 증상을 나타냄을 확인함으로써 뇌전증 동물 모델로서 활용될 수 있다고 판단하였다.In addition, the seizure symptom induced by pentylenetradazole (PTZ) injection at 30 mg / kg was similar to that caused by KA. As shown in FIG. 4C, the time until the appearance of the first seizure in the Kcnk1-deficient mouse was remarkably shorter than that in the control group, and in the seizure symptom analysis by the number of injections, the seizure symptoms were provoked by a small number of injections as shown in FIG. Respectively. These results suggest that Kcnk1 deficient mice can be used as an animal model of epilepsy by confirming that they exhibit symptoms of arthropathy more easily than the control group.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
본 발명에 따른 CRISPR/Cas9 유전자가위에 의하여 Kcnk1 유전자가 결손된 마우스는 우울증 및/또는 뇌전증의 진행 경과와 병리학적 기전 연구의 동물 모델로 유용하게 이용될 수 있으며, 또한 우울증 및/또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝에 유용하게 이용될 수 있을 것으로 기대된다.The mouse lacking the Kcnk1 gene by the CRISPR / Cas9 gene scissors according to the present invention can be used as an animal model of progression of depression and / or brain metastasis and pathological mechanism studies, and also can be used as an animal model of depression and / And can be usefully used for screening candidate drugs for prevention or treatment.

Claims (7)

  1. Kcnk1(Potassium Channel Subfamily K) 유전자가 결손(knock-out)된 마우스인 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델.An animal model of depression or epilepsy, characterized in that the Kcnk1 (Potassium Channel Subfamily K) gene is a knock-out mouse.
  2. Kcnk1 유전자 중 exon1 일부를 표적으로 하여 CRISPR/Cas9 유전자가위로 염색체 상에서 제거한 Kcnk1 유전자 결손 마우스를 얻는 단계;Obtaining a Kcnk1 gene-deficient mouse in which CRISPR / Cas9 gene has been removed from its chromosome by targeting a part of exon 1 of Kcnk1 gene;
    상기 Kcnk1 유전자 결손 마우스 간에 교배를 통하여 2세대 마우스를 얻는 단계; 및Obtaining a second-generation mouse through cross-breeding among the Kcnk1 gene-deficient mice; And
    상기 2세대 마우스로부터 Kcnk1 유전자가 발현되지 않는 마우스를 선별하는 단계;Selecting a mouse from which the Kcnk1 gene is not expressed from the second generation mouse;
    를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 동물 모델의 제조방법.≪ / RTI > wherein the method comprises the step of administering to a mammal an animal model of depression or epilepsy.
  3. 제1항의 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;Administering a candidate drug to the depressive or epileptic animal model of claim 1;
    상기 후보약물 투여 후 상기 동물 모델의 발작 여부 및 생존율을 측정하는 단계; 및Measuring the seizure frequency and survival rate of the animal model after administration of the candidate drug; And
    상기 후보약물을 투여하지 않은 대조군과 비교하여 발작을 완화시켜주거나 생존율을 향상시키는 후보약물을 선별하는 단계;Selecting a candidate drug that alleviates the seizure or improves the survival rate as compared with the control group in which the candidate drug is not administered;
    를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug is selected from the group consisting of: < RTI ID = 0.0 > (I) < / RTI >
  4. 제1항의 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;Administering a candidate drug to the depressive or epileptic animal model of claim 1;
    상기 후보약물 투여 후 상기 동물 모델의 행동학적 특성을 분석하는 단계; 및Analyzing the behavioral characteristics of the animal model after administration of the candidate drug; And
    상기 후보약물을 투여하지 않은 대조군과 비교하여 우울증 증상을 완화시키는 행동학적 특성을 보이는 후보약물을 선별하는 단계;Selecting a candidate drug exhibiting a behavioral characteristic that alleviates the symptoms of depression as compared with a control group to which the candidate drug is not administered;
    를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug is selected from the group consisting of: < RTI ID = 0.0 > (I) < / RTI >
  5. 제1항의 우울증 또는 뇌전증 동물 모델에 후보약물을 투여하는 단계;Administering a candidate drug to the depressive or epileptic animal model of claim 1;
    상기 후보약물 투여 후 상기 동물 모델의 별아교세포에서 수동전도도를 측정하는 단계; 및Measuring passive conductance in astrocytes of the animal model after administration of the candidate drug; And
    상기 후보약물을 투여하지 않은 대조군과 비교하여 수동전도도를 증가시키는 후보약물을 선별하는 단계;Selecting a candidate drug that increases passive conductivity compared to a control to which the candidate drug is not administered;
    를 포함하는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug is selected from the group consisting of: < RTI ID = 0.0 > (I) < / RTI >
  6. 제3항 내지 제5항 중 어느 한 항에 있어서,6. The method according to any one of claims 3 to 5,
    상기 후보약물은 우울증 또는 뇌전증 예방 또는 치료제인 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug is a drug for the prevention or treatment of depression or brain metastasis.
  7. 제4항에 있어서,5. The method of claim 4,
    상기 후보약물은 대조군의 인지 또는 학습 능력에 유의적인 영향을 미치지 않는 것을 특징으로 하는, 우울증 또는 뇌전증 예방 또는 치료용 후보약물의 스크리닝 방법.Wherein the candidate drug has no significant effect on the cognitive or learning ability of the control group.
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