KR100734815B1 - 24 Transgenic dementia mice expressing htau24 and the method for preparing thereof - Google Patents
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Abstract
Description
도 1은 본 발명에 따른 htau24 유전자가 삽입된 형질전환 마우스에 있어서, pNSE/htau24 유전자의 삽입을 확인하기 위하여 서던 블럿(Southern blot)을 실시한 결과이다. 여기에서, A는 재조합 융합 유전자 pNSE/htau24 플라스미드의 구조를, B 및 C는 삽입유전자의 동정결과를 각각 나타낸다.1 is a result of performing Southern blot in order to confirm the insertion of the pNSE / htau24 gene in the transgenic mouse inserted htau24 gene according to the present invention. Here, A represents the structure of the recombinant fusion gene pNSE / htau24 plasmid, and B and C represent the identification result of the insert gene, respectively.
도 2는 본 발명에 따른 NSE/htau24 유전자가 각각 삽입된 형질전환 치매마우스 및 동일 연령표준(age-matched) 대조군 마우스의 수중 미로검사(Water Maze Tests) 결과이다.Figure 2 shows the results of the Water Maze Tests of transgenic demented mice and age-matched control mice inserted with NSE / htau24 genes according to the present invention, respectively.
도 3은 본 발명에 따른 NSE/htau24 유전자가 각각 삽입된 형질전환 치매 마우스 뇌 조직에서 tau의 과다발현과 tau 결합단백질의 과인산화, 그리고 PHFs의 생성을 웨스턴블랏 및 전자현미경으로 확인한 결과이다.3 is a result of Western blot and electron microscopy confirming the overexpression of tau, the overphosphorylation of tau binding protein, and the production of PHFs in transgenic demented mouse brain tissues in which the NSE / htau24 gene is inserted according to the present invention.
도 4는 본 발명에 따른 NSE/htau24 유전자가 각각 삽입된 형질전환 치매 마우스의 tau 결합 단백질의 과인산화를 연령별로 웨스턴 블랏을 확인한 결과이다.4 is a result of Western blot analysis of the hyperphosphorylation of tau binding proteins of transgenic dementia mice in which NSE / htau24 genes are inserted according to the present invention.
도 5는 본 발명에 따른 NSE/htau24 유전자가 삽입된 형질전환 치매 마우스의 뇌 조직에서 리튬 투여에 의한 tau 결합단백질 인산화 저해를 웨스턴 블랏 확인한 결과이다.Figure 5 is the result of Western blot confirmed the inhibition of tau binding protein phosphorylation by lithium administration in the brain tissue of the transgenic demented mouse inserted with the NSE / htau24 gene according to the present invention.
본 발명은 APP/htau24 유전자를 발현하는 형질전환 치매 마우스에 관한 것으로, 보다 구체적으로는 뇌특이적 NSE(Neuron-Specific Enolase) 유전자의 프로모터(promoter)를 인간 유래의 htau24 치매 유발 유전자에 연결시켜 제조한 융합(hybrid) 유전자인 NSE/htau24를 마우스의 수정란에 미세주입(microinjection)하고, 이 수정란을 대리모 마우스의 난관(oviduct)에 이식함으로써 생산된 형질전환 치매 마우스에 관한 것이다.The present invention relates to a transgenic dementia mouse expressing the APP / htau24 gene, and more particularly, by connecting a promoter of the brain-specific Neuron-Specific Enolase (NSE) gene to a htau24 dementia causing gene derived from humans. It relates to a transgenic dementia mouse produced by microinjecting a hybrid gene, NSE / htau24, into a fertilized egg of a mouse and implanting the fertilized egg into an oviduct of a surrogate mother mouse.
치매(dementia)는 기억력 장애, 판단력 상실 등 전신기능의 전반적인 장애를 동반하는 질환이다. 치매의 발병 형태는 매우 다양한데, 약 50% 정도는 알츠하이머형 치매(dementia of Alzheimer type), 20∼30%는 혈관성 치매(dementia of Vascular type)이고, 알코올성 치매 및 파킨슨병 치매 등이 있으며, 약 15∼20%는 알츠하이머형 및 혈관성 치매 양쪽성으로 발병하는 것으로 알려져 있다.Dementia is a disorder accompanied by general disorders of systemic function, such as memory impairment and loss of judgment. There are many forms of dementia, about 50% of which are dementia of Alzheimer type, 20 to 30% of dementia of Vascular type, alcoholic dementia and Parkinson's dementia. It is known that ˜20% develop both Alzheimer's and vascular dementia.
이 중 치매의 가장 중요한 발병 형태인 알츠하이머 질환(Alzheimer disease; AD)은 50세 이전에는 증상이 나타나는 경우가 드물지만 60세 이후로는 발생 빈도가 점진적으로 증가하는 노인성 치매질환으로서, 의학기술의 발전 및 삶의 질 향상으로 인한 노인 인구의 증가에 따라 우리나라뿐만 아니라 전세계적으로 발병 인구가 급속히 증가하고 있는 질환이다. 우리나라에서 1995년 65세 이상으로 등록된 알츠 하이머 질환 환자수는 241,000명으로 노인 전체 인구의 8.3%를 차지하고 있으며, 2020년에는 619,000명이 될 것으로 예측된 바 있다[보건사회연구원(1995), 후생일보(제 4763) 1998년 12월 28일]. 알츠하이머 질환은 초기에 진단되는 경우에 한해서만 제한적으로 치료가 가능하기 때문에 이를 조기에 정확하게 진단할 수 있는 방법 및 치료 방법의 개발이 절실히 요구되고 있으나, 이에 대한 발병원인조차 정확하게 밝혀지지 않은 실정이다.Alzheimer's disease (AD), the most important form of dementia, is an elderly dementia disease that occurs rarely before
알츠하이머 질환의 원인은 아직 확실하게 규명되어 있지 않으나 다음 2가지 단백질의 기능이 주목을 받고 있다. 첫째는 아밀로이드 β-39/43(Amyloid β-39/ 43)이라고 하는 신경 독성 단백질인데, 이것이 고농도로 농축되어 플라그(plague)를 형성, 뇌의 기억 및 인식을 담당하는 신경세포 부위에 침적되어 뉴런의 DNA에 상해를 일으켜 알츠하이머를 일으킨다는 것이다. 또 하나는 토(tau)라는 실처럼 생긴 가느다란 단백질인데, 이것이 서로 꼬여서 엉킨 모양의 수초(tangle)를 형성하여 단백질이 이상적으로 과인산화됨에 따라 뇌신경 세포의 사멸이 초래된다는 것이다.The cause of Alzheimer's disease is not yet well understood, but the function of the following two proteins is attracting attention. The first is a neurotoxic protein called amyloid β-39 / 43, which is concentrated at high concentrations to form plaques, which are deposited on nerve cell sites responsible for brain memory and recognition. That damages the DNA of Alzheimer's. The other is a thin, thread-like protein called tau, which twists together to form a tangled tangle, resulting in the death of neuronal cells as the protein is ideally overphosphorylated.
이 중, AD 질환의 핵심 원인 물질로서 최근 주목을 받고 있는 것이 tau 단백질인데 tau의 과인산화에 의해서 PHF가 생성되는 것이다. 이러한 PHF는 NFT(Neuron Fibrillary Tangles)를 구성하여 뉴론의 퇴행성에 중요한 역할을 하는 것으로 알려져 있다.Among them, tau protein, which is attracting attention recently as a key cause of AD disease, is PHF produced by tau hyperphosphorylation. These PHFs are known to play an important role in neuron degeneration by forming Neuron Fibrillary Tangles (NFT).
이러한 치매의 원인을 밝히고, 치료 물질을 개발하기 위해서는 임상실험을 하기 전에 실제로 실험을 할 수 있는 치매 동물 모델의 개발이 필수적이다. 이를 위하여, Thy 1,2 조절하의 htau40 및 htau24의 치매 실험쥐가 개발되어 이용된 바 있으나, 상기 설명한 바와 같은 NSE 조절하의 htau24 유전자가 삽입되어 치매가 유발된 치매 실험쥐는 아직까지 개발된 바 없으며 NFT의 생성을 발현하지 못하였다. In order to identify the cause of such dementia and develop a therapeutic substance, it is essential to develop a dementia animal model that can be actually tested before the clinical trial. For this purpose, dementia mice under the control of
이에 본 발명자들은 상기와 같은 기술현실을 파악하고 알츠하이머 치매 질환의 진단 및 치료를 위한 동물 모델을 개발하기 위하여 연구한 결과, 치매 유발 유전자인 htau24 유전자를 도입시킨 형질전환 치매 마우스를 개발할 수 있었다. 즉, 동물 체내에서 발현되어 치매의 원인이 되는 독성 단백질 중 하나인 tau의 과인산화 생성을 증가시키는 APP/htau24 유전자를 NSE 프로모터에 결합시키고 이러한 융합 유전자를 마우스의 수정란에 주입시킨 수정란을 착상시켜 생산한 형질전환 치매 마우스가 치매 환자에게서 관찰되는 병리학적 특징을 나타냄을 발견하고 본 발명을 완성하였다.Therefore, the present inventors have been able to develop a dementia mouse incorporating htau24 gene, which is a dementia-inducing gene, as a result of studying the technical reality as described above and developing an animal model for diagnosing and treating Alzheimer's dementia disease. In other words, APP / htau24 gene, which is expressed in the animal and increases the superphosphorylation of tau, one of the toxic proteins that cause dementia, is coupled to the NSE promoter and implanted into a fertilized egg of a mouse. One transgenic dementia mouse was found to exhibit the pathological features observed in patients with dementia and completed the present invention.
따라서, 본 발명의 목적은 NSE/htau24 유전자를 발현하는 형질전환 치매 마우스를 제공하는 것이다.It is therefore an object of the present invention to provide a transgenic dementia mouse expressing the NSE / htau24 gene.
상기한 본 발명의 특징 및 그 외의 다른 특징들은 후술하는 본 발명의 상세한 설명에 의하여 당업자에게 잘 드러날 수 있다.The above-described features of the present invention and other features can be well seen by those skilled in the art by the following detailed description of the present invention.
상기한 목적을 달성하기 위하여, 본 발명에서는 뇌특이적 NSE(Neuron-Specific Enolase) 유전자의 프로모터(promoter)와 인간 htau24 치매 유전자를 연결시켜 융합 유전자 NSE/htau24를 제조한 다음, 상기 융합 유전자를 마우스의 수정 란에 미세주입하고, 이 수정란을 대리모 마우스의 난관에 이식함으로써 형질전환된 치매 마우스를 제조함을 그 특징으로 한다.In order to achieve the above object, in the present invention, a promoter of the brain-specific Neuron-Specific Enolase (NSE) gene and a human htau24 dementia gene are linked to produce a fusion gene NSE / htau24, and then the fusion gene is mouse. It is characterized by producing a transformed demented mouse by micro-injecting the fertilized egg of and implanting it into the fallopian tube of the surrogate mother mouse.
본 발명은 형질전환 치매 마우스로서, 그의 크로모좀 내에 NSE/htau24 유전자가 삽입되어 있고, 이 유전자는 뇌특이적 NSE 프로모터에 기능적으로 연결되어 있어, 상기 htau24 유전자가 치매를 유발하는 수준으로 발현되어, 그 결과 행동이상이 발현되는 치매 모델 동물로서의 형질전환 치매 마우스를 제공함을 그 특징으로 한다.The present invention is a transgenic dementia mouse, in which the NSE / htau24 gene is inserted into its chromosome, and this gene is functionally linked to a brain-specific NSE promoter, whereby the htau24 gene is expressed at a level causing dementia, As a result, it provides a transgenic dementia mouse as a dementia model animal in which behavioral abnormalities are expressed.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
정상인의 뇌에 존재하는 tau 단백질은 대개 정상레벨에서 인산화되어 존재하게 되나, 알츠하이머 환자의 뇌에서는 tau 및 tau-결합단백질이 이상적으로 활성화되어가는 과정에서 PHFs로 구성되는 NFTs가 다량으로 생산된다. 알츠하이머 질환은 이렇게 생성된 NFTs가 뇌 영역 중 기억 또는 인식을 담당하는 뉴런에 고농도로 장기간 축적되면서 DNA에 상해를 일으켜 발생하는 것으로 알려져 있다. 알츠하이머 환자의 뇌에서는 tau 유전자에 의하여 생산이 증가된 이상적 과다인산화에 의하여 행동이상의 발현이 촉진되고, GSK3β 및 이의 결합단백질 발현이상활성이 초래된다. NFTs는 뇌에서 기억과 인식을 담당하는 뉴런에 손상을 입히는 독성 단백질이며 기억장애를 초래한다.The tau proteins present in normal brains are usually phosphorylated at normal levels, but in Alzheimer's brains, NFTs composed of PHFs are produced in large quantities during the ideal activation of tau and tau-binding proteins. Alzheimer's disease is known to be caused by DNA damage caused by long-term accumulation of NFTs in high concentrations in neurons responsible for memory or cognition in the brain region. In the brains of Alzheimer's patients, ideal hyperphosphorylation with increased production by the tau gene promotes the expression of behavioral abnormalities, resulting in GSK3β and its associated protein dysregulation. NFTs are toxic proteins that damage the neurons responsible for memory and cognition in the brain and cause memory disorders.
본 발명에서는 상기와 같은 NSE/htau24 유전자와 NSE 프로모터가 서로 결합된 융합 유전자 NSE/htau24를 인간을 제외한 각종 동물에 도입할 수 있다. 이러한 동물로는 마우스(mouse) 또는 랫드(rat) 등을 들 수 있으나 랫드는 모델 동물을 만 들기 어려우므로 바람직하게는 마우스를 이용하는 것이 좋다.In the present invention, the fusion gene NSE / htau24 in which the NSE / htau24 gene and the NSE promoter are coupled to each other may be introduced into various animals except humans. Such animals include a mouse or a rat, but rats are difficult to make model animals, and therefore, it is preferable to use a mouse.
본 발명에서 NSE/htau24 유전자와 NSE 프로모터가 결합된 융합 유전자는 당업계에 주지된 각종 형질전환 방법에 따라 동물의 크로모좀 내로 삽입시킬 수 있다. 이러한 형질전환 방법의 예를 들면, 동물의 수정란에 융합 유전자를 미세주입하는 방법 또는 레트로바이러스 벡터를 이용하는 방법 등을 들 수 있다. 또는 상기 재조합 융합 유전자를 수정란에 이식시킨 수정란을 다른 대리모 실험쥐에 착상시키는 방법을 이용할 수도 있다. 그러나, 실험을 하는 조작자의 안전성 측면에서, 융합 유전자를 수정란에 미세주입하는 방법이 보다 바람직하다.In the present invention, the fusion gene in which the NSE / htau24 gene and the NSE promoter are combined may be inserted into an animal chromosome according to various transformation methods well known in the art. Examples of such transformation methods include a method of microinjecting a fusion gene into a fertilized egg of an animal or a method using a retroviral vector. Alternatively, a method of implanting a fertilized egg transplanted with the recombinant fusion gene into a fertilized egg may be used in another surrogate mother mouse. However, from the viewpoint of the safety of the experimenter, the method of microinjecting the fusion gene into the fertilized egg is more preferable.
본 발명의 하기 제조예에서는 상기와 같은 치매 유발 유전자인 인간 NSE/htau24 유전자와 NSE 프로모터가 각각 결합된 재조합 융합 유전자 NSE/htau24를 BDF1 마우스의 수정란에 미세주입하고, 이와 같이 유전자가 주입된 수정란을 ICR 마우스로 이식하여 형질전환 치매 마우스 새끼를 생산하였다.In the following preparation example of the present invention, the recombinant fusion gene NSE / htau24, in which the human NSE / htau24 gene and the NSE promoter, respectively, are combined as described above is microinjected into the fertilized egg of BDF1 mouse, and the fertilized egg injected with the gene as described above. Transgenic demented mouse pups were produced by transplantation into ICR mice.
상기와 같이 NSE/htau24 유전자와 NSE 프로모터가 기능적으로 결합된 융합 유전자가 삽입된 형질전환 치매 마우스는 htau24 유전자가 NSE 프로모터에 의하여 발현됨으로써 알츠하이머로 인한 다양한 병리현상을 모두 나타내는 치매 유발 마우스 모델이다. 즉, 상기 제조된 NSE/htau24 유전자를 발현하는 형질전환 치매 마우스를 각각 번식시켜 알츠하이머의 표현형(phenotype)을 측정한 결과, 치매 환자에게서 특징적으로 나타나는 병리현상으로 tau 및 tau-결합단백질의 이상적 인산화와PHFs가 확인되었으며, 수중 미로 검사(Water Maze Test)를 통하여 뇌기능에도 이상이 있음이 확인되었다. 이를 통하여, 본 발명에 따른 형질전환 치매 마우스는 치 매 환자와 매우 유사한 행동 장애를 보이는 것을 알 수 있었다.As described above, the transgenic dementia mouse into which the fusion gene in which the NSE / htau24 gene and the NSE promoter are functionally inserted is a dementia-induced mouse model that exhibits various pathologies caused by Alzheimer's disease because the htau24 gene is expressed by the NSE promoter. In other words, as a result of measuring the phenotype of Alzheimer's disease by breeding transgenic dementia mice expressing the prepared NSE / htau24 gene, ideal phosphorylation of tau and tau-binding proteins as a pathological phenomenon characteristic of dementia patients. PHFs were identified, and abnormal water function was confirmed through the Water Maze Test. Through this, the transgenic dementia mouse according to the present invention was found to exhibit a behavioral disorder very similar to the dementia patients.
한편, 본 발명에 따른 형질전환 치매 마우스를 정상 마우스와 교배하는 경우, NSE/htau24 유전자가 그 다음 세대로 전달된다. 즉, 교배에 의하여 상기 NSE/htau24 유전자는 유전이 가능하다.On the other hand, when the transgenic demented mouse according to the present invention is crossed with normal mice, the NSE / htau24 gene is transferred to the next generation. In other words, the NSE / htau24 gene can be inherited by crossing.
본 발명에 따른 형질전환 치매 마우스는 치매 환자가 나타내는 것과 유사한 행동이상을 나타내며, tau 및 tau-결합단백질의 이상적 인산화가 관찰되는 등 거의 완벽한 치매 증상을 보이는 것이 특징이다. 따라서, 상기와 같은 치매 관련성 물질의 발현 증가와 같은 표현 형질을 이용하는 경우 치매 발병 매커니즘의 연구에 매우 유용하게 이용할 수 있으며, 특히 치매의 치료제 또는 조기 진단법의 개발 등에 유용하게 이용될 수 있다. 현재 국내·외에서 개발되었거나 개발중인 치매 치료제를 투여하여 인체와 유사한 조건 하에서 나타나는 다양한 치매 치료 효과, 즉 아밀로이드 β-42 축적의 억제, 치매로 인한 행동 이상의 완화, 및 GSK3β와 이의 결합단백질 활성의 저하를 확인함으로써 치매 치료제의 치매 치료 효과를 검정할 수 있으며, 또한 치매 질환의 원인을 연구하는 동물 모델로도 사용이 가능하다.Transgenic demented mice according to the present invention exhibit behavioral abnormalities similar to those exhibited by patients with dementia, and are characterized by almost complete dementia symptoms such as ideal phosphorylation of tau and tau-binding proteins. Therefore, in the case of using the expression traits such as the increased expression of the dementia-related substances, it can be very useful for the study of the mechanism of dementia, and particularly useful for the development of a therapeutic or early diagnosis of dementia. The treatment of dementia currently developed or developed at home and abroad is used to treat various dementia treatment effects under conditions similar to those of the human body, such as inhibiting amyloid β-42 accumulation, alleviating behavioral abnormalities due to dementia, and decreasing GSK3β and its binding protein activity. By confirming, the dementia treatment effect of the dementia treatment agent can be tested, and it can also be used as an animal model for studying the cause of dementia disease.
이하, 본 발명의 구성을 실시예 및 시험예를 들어 보다 구체적으로 설명하지만, 본 발명의 권리범위가 이들에 한정하는 것은 아니다.Hereinafter, the structure of the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited thereto.
[제조예 1] NSE/htau24 유전자가 삽입된 형질전환 치매 마우스의 제조Preparation Example 1 Preparation of Transgenic Dementia Mice Inserted with the NSE / htau24 Gene
① 유전자의 확보 및 재조합① Gene acquisition and recombination
본 실험에 사용된 유전자 중 NSE 프로모터는 Sutcliffe 박사(Research Institute of Scrippe Clinic)로부터, RT-PCR 방법으로 사람의 세포주에서 얻어 사 용하였다.Among the genes used in this experiment, the NSE promoter was obtained from Sutcliffe Ph.D. (Research Institute of Scrippe Clinic) and obtained from human cell lines by RT-PCR method.
유전자의 재조합은 서열번호 5의 NSE 프로모터, 서열번호 6의 htau24 유전자 및 종결인자의 순서대로 결합한 다음 특이적 프라이머[센스:5´-GCACT AGTCA GGTGA ACTTT GAACC AGGAT G-3´(서열번호 1), 안티센스:5´-GCACT AGTCT GATCA CAAAC CCTGC TTG-3´(서열번호 2)]를 이용하여 94℃에서 30초간 변성, 62℃에서 30초간 어닐링 및 72℃에서 45초간 합성하는 사이클을 25회 수행하여 PCR 증폭하였다. pGEM-T에 클로닝하여 XhoI/EcoRI으로 절단된 pNSE-splice 벡터에 삽입하여 완성하였다.Recombination of the gene was performed by combining the NSE promoter of SEQ ID NO: 5, the htau24 gene of SEQ ID NO: 6, and the terminator, followed by specific primers (sense: 5'-GCACT AGTCA GGTGA ACTTT GAACC AGGAT G-3 '(SEQ ID NO: 1)). Antisense: 5′-GCACT AGTCT GATCA CAAAC CCTGC TTG-3 ′ (SEQ ID NO: 2)], followed by 25 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 62 ° C. for 30 seconds, and synthesis at 45 ° C. for 45 seconds. PCR amplification. Cloning into pGEM-T was completed by insertion into a pNSE-splice vector digested with XhoI / EcoRI.
② 융합 유전자의 순수분리 및 마우스 수정란에 미세주입② Fine injection of fusion gene and injection into mouse fertilized egg
서열번호 7의 재조합 융합 유전자(recombinat fusion gene) NSE/htau24를 순수분리하기 위하여 박테리아에서 기원된 유전자 부위를 절단하여 제거하였고, 진핵세포 발현 부위만을 수확하여 물에 녹여 사용하였다. 순수분리된 상기 융합 유전자 NSE/htau24를 4ng/㎕의 농도로 희석하여 마우스(BDF1)의 수정란에 미세주입하였다. 상기 유전자가 주입된 수정란을 가임신한 ICR 마우스에 착상시켜 새끼를 생산하였다.Recombinat fusion gene (SEQ ID NO: 7) NSE / htau24 was purified by cleaving a gene region derived from bacteria for pure separation, and only eukaryotic expression sites were harvested and dissolved in water. The purely isolated fusion gene NSE / htau24 was diluted to a concentration of 4 ng / μl and injected into the fertilized eggs of mice (BDF1). Embryos implanted with the gene were implanted into ICR mice of fertility to produce offspring.
③ 형질전환 마우스의 확인③ Identification of Transgenic Mouse
상기에서 생산된 새끼의 꼬리를 절단하여 게놈 DNA를 분리한 후, 상기 재조합 융합 유전자에 특이적인 프라이머(Gibco BRL Co. 제작의뢰)[센스: 5´-CTGCAGCTCTTGACTCATCG-3´(서열번호3), 안티센스: 5´-GCACTAGTCTGATCACAAACCCTGCTTG-3´(서열번호4)]를 이용하여 94℃에서 30초간 변성, 62℃에서 30초간 어닐링 및 72℃에서 45초간 합성하는 사이클을 25회 반복하는 DNA-PCR을 수행한 다음, 서던 블랏 하이브리디제이션(Southern blot hybridization)을 실시하여 NSE/htau24 유전자의 삽입을 확인하였으며, 그 결과를 도 1에 나타내었다.After cutting the tail of the produced offspring to isolate genomic DNA, primers specific for the recombinant fusion gene (Gibco BRL Co. production request) [Sense: 5 '-CTGCAGCTCTTGACTCATCG-3' (SEQ ID NO: 3), antisense : DNA-PCR was performed using 5'-GCACTAGTCTGATCACAAACCCTGCTTG-3 '(SEQ ID NO: 4)] for 25 seconds of denaturation at 94 ° C for 30 seconds, annealing at 62 ° C for 30 seconds, and 45 seconds at 72 ° C. Next, Southern blot hybridization was performed to confirm the insertion of the NSE / htau24 gene, and the results are shown in FIG. 1.
플라스미드 pNSE/htau24는 NSE 유전자 프로모터의 조절 하에 위치하고 있는 htau24 유전자를 포함하고 있으며(도 1A). 또한 DNA-PCR 및 서던블랏 분석 결과, NSE/htau24 트랜스 유전자를 갖는 형질전환 생쥐에서 1,112bp 크기의 PCR 7.5kb의 서던블랏 산물이 확인되었다(도 1B의 #7424).Plasmid pNSE / htau24 contains the htau24 gene located under the control of the NSE gene promoter (FIG. 1A). In addition, DNA-PCR and Southern blot analysis revealed a Southern blot product of 7.5 kb PCR having a size of 1,112 bp in transgenic mice carrying the NSE / htau24 trans gene (# 7424 in FIG. 1B).
또한, 상기와 같이 htau24 유전자가 확인된 형질전환 마우스를 정상의 마우스와 교배한 결과, htau24 유전자가 후대로 전달됨을 확인할 수 있었다.In addition, as a result of crossing the mouse with a transgenic mouse identified with the htau24 gene as described above, it was confirmed that the htau24 gene is transferred later.
상기와 같이, NSE/htau24 융합 유전자를 진핵세포 발현부위로 삽입시켜 형질전환시킨 마우스의 수정란, 즉 NSE/htau24 유전자가 크로모좀 내로 삽입된 형질전환 치매 마우스의 수정란을 국제기탁기관인 한국생명공학연구소 유전자은행(Korean Collection for Type Cultures)에 2006년 2월 2일자로 기탁하였으며, 그로부터 기탁번호 KCTC 10906BP를 부여받았다.As described above, a fertilized egg of a mouse transformed by inserting an NSE / htau24 fusion gene into an eukaryotic cell expression site, that is, a fertilized egg of a transgenic demented mouse in which an NSE / htau24 gene is inserted into a chromosome, is a Korea Deposit Research Institute gene. It was deposited with the Bank of Korea (Korean Collection for Type Cultures) on February 2, 2006, and was given accession number KCTC 10906BP.
[시험예 1] 형질전환 치매 마우스의 각 조직에서의 NSE/htau24 유전자의 발현 확인과 tau 및 tau-결합단백질의 과인산화[Test Example 1] Confirmation of NSE / htau24 gene expression and hyperphosphorylation of tau and tau-binding proteins in tissues of transgenic dementia mice
상기 형질전환 치매 마우스로부터 각 조직을 분리하여 NSE/htau24 유전자가 조직 특이적으로 발현하는지를 확인하였다. 이를 위하여, 상기 형질전환 치매 마우스의 뇌, 간 그리고 신장을 이용하여 랫드 NSE 프로모터 조절하의 htau24 유전자의 각 조직에서의 발현을 RT-PCR로 확인하였다. 상기 RT-PCR은 94℃에서 30초간 변성, 62℃에서 30초간 어닐링 및 72℃에서 45초간 합성하는 사이클을 28회 반복한 후, 72℃에서 7분간 처리한 것이다.Each tissue was isolated from the transgenic demented mouse to confirm whether the NSE / htau24 gene was expressed in a tissue-specific manner. To this end, the brain, liver and kidney of the transgenic demented mouse was confirmed by RT-PCR expression in each tissue of the htau24 gene under the control of rat NSE promoter. The RT-PCR is denatured at 94 ° C. for 30 seconds, annealed at 62 ° C. for 30 seconds, and synthesized for 45 seconds at 72 ° C. after 28 cycles, followed by treatment at 72 ° C. for 7 minutes.
그 결과를 (도 3A)에 나타내었다. 대조군으로서의 β-액틴 시그널 및 그들의 전사체(transcript, 492 bp)를 RNA의 로딩을 가리키기 위하여 나타내었다. NSE/htau24 융합 유전자의 발현은 뇌-특이적 방식(brain-specific manner)으로 조절되었다.The results are shown in (FIG. 3A). Β-actin signals and their transcripts (492 bp) as controls were shown to indicate the loading of RNA. Expression of the NSE / htau24 fusion gene was regulated in a brain-specific manner.
Tau 단백질의 과다발현으로 인한 tau 및 tau-결합단백질의 이상적 과인산화를 웨스턴 블랏으로 확인하였다. 그 결과를 (도 3B)에 나타내었다.The ideal hyperphosphorylation of tau and tau-binding proteins due to overexpression of the Tau protein was confirmed by Western blot. The result is shown in (FIG. 3B).
Tau 단백질의 과다발현으로 tau 및 tau-결합단백질의 과인산화 및 PHFs의 생성을 촉진하였다. (도 3C, 도 3D)Overexpression of the Tau protein promoted hyperphosphorylation of tau and tau-binding proteins and production of PHFs. (FIG. 3C, FIG. 3D)
[시험예 2] 뇌 기능 이상의 측정Test Example 2 Measurement of Brain Function Abnormalities
상기 NSE/htau24 형질전환 치매 마우스(18개월령)가 정상적인 쥐와 비교했을 때 뇌 기능에 이상이 있는지의 여부를 확인하기 위하여 수중 미로 검사(Water Maze Test)를 실시하였다.The NSE / htau24 transgenic dementia mouse (18 months old) was subjected to a water maze test (Water Maze Test) to check whether there is any abnormality in brain function compared to normal mice.
그 결과를 도 2에 나타내었다. 도 2는 수중 미로 검사에서 전방 플랫폼 위치를 가로지르는 도피 지연(escape latency)(도 2A), 도피 거리(escape distance)(도 2B) 및 수영 속도(swimming velocity)(도 2C)의 패턴을 측정한 결과이다[평균ㅁ3회 실험의 SD, 수중 미로 검체와 수행된 3회 실험의 메디안 지수(median values) 및 SD는 n=3 P<0.01이다]. 형질전환 치매 마우스의 도피 지연 및 도피 거리는 대조군 마우스에 비하여 더 길게 나타나는 일관적인 경향을 보였다. 그러나, 수영 속도에 있어서는 형질전환 마우스와 대조군 마우스가 유사한 수준을 보였다. The results are shown in FIG. FIG. 2 measures the pattern of escape latency (FIG. 2A), escape distance (FIG. 2B) and swimming velocity (FIG. 2C) across the front platform position in an underwater maze test. Results [mean SD of mean 3 experiments, maze specimens in water and median values and SD of 3 experiments performed n = 3 P <0.01]. Escape delays and escape distances of transgenic demented mice showed a consistent tendency to appear longer than control mice. However, transgenic mice and control mice showed similar levels in swimming speed.
상기 결과에서 알 수 있는 바와 같이, NSE/htau24 형질전환 치매 마우스와 정상 쥐 사이에서 도피 지연 및 도피 거리 측정치의 현저한 차이가 나타났으며, 결과적으로 본 발명에 의한 형질전환 치매 마우스는 뇌 기능의 이상으로 인하여 행동에 이상이 나타남을 확인하였다.As can be seen from the above results, there was a significant difference in escape delay and escape distance measurements between NSE / htau24 transgenic dementia mice and normal mice. As a result, the transgenic demented mice according to the present invention had abnormal brain function. As a result, it was confirmed that the abnormal behavior appeared.
[시험예 3] 형질전환 치매 마우스의 뇌에서의 연령별 과인산화증가Experimental Example 3 Hyperphosphorylation Increased in the Brain of Transgenic Dementia Mice
형질전환 치매 마우스의 뇌에서 치매의 원인이 되는 독성 단백질인 tau의 이상적 과인산화가 증가하였는지를 웨스턴 블랏(western으로 blot)으로 확인하였다(도 4).In the brain of transgenic demented mice, it was confirmed by Western blot that the ideal hyperphosphorylation of tau, a toxic protein that causes dementia, increased (FIG. 4).
형질전환 마우스 및 대조군 마우스의 뇌로부터 분리한 단백질 40㎍을 전이시킨 니트로셀룰로스 필터를 항-인간tau, p-tau, GSK3β, p-GSK3β, β-칼리닌(β-calinin), 그리고 p-β-칼리닌 항체들과 함께 배양하고 그 수준을 정량화하였다(도 4A). 각각의 수치는 3회 실험 결과를 평균±SD 로 나타낸 것이다(P<0.028). Nitrocellulose filters, which transferred 40 μg of protein isolated from the brains of transformed and control mice, were treated with anti-human tau, p-tau, GSK3β, p-GSK3β, β-calinin, and p-β. -Incubated with kalinin antibodies and quantified their levels (FIG. 4A). Each figure represents the mean ± SD of three experiments (P <0.028).
도 5는 형질전환 마우스 및 연령 표준 대조군 마우스에 리튬을 농도별로 처리한 후 뇌 단백질 추출물을 항-GSK3β와 항 pGSKβ, β-칼리닌, tau와 p-tau 그리고 PS1-CTF로 보온한 다음, 항-래빗-HRP 구체화(visualization)하여 tau 결합단백질 인산화 저해를 웨스턴 블랏으로 확인한 결과이다. 대조군 마우스 p-GSK3β 활성의 증가가 관찰되었다(도 5B). 상기 결과로부터 본 발명의 형질전환 치매 마우스에서 tau 결합단백질의 인산화가 더욱 활성화되었음을 확인하였다.Figure 5 after transgenic mice and age standard control mice treated with lithium by concentration, brain protein extracts were insulated with anti-GSK3β and anti-pGSKβ, β-kalinin, tau and p-tau and PS1-CTF, and then Rabbit-HRP visualization to confirm tau binding protein phosphorylation inhibition by Western blot. An increase in control mouse p-GSK3β activity was observed (FIG. 5B). From the above results, it was confirmed that phosphorylation of tau binding protein was more activated in the transgenic dementia mouse of the present invention.
이상에서 설명한 바와 같이, 본 발명에서는 치매 유발 유전자인 NSE/htau24 유전자와 NSE 프로모터를 결합시킨 융합 유전자를 마우스 수정란에 주입하여 형질전환 치매 마우스를 제조하였으며, 본 발명에 의한 치매 마우스는 NES/htau24 유전자가 과다 발현함에 따라 치매 환자의 행동이상을 그대로 나타내며, 특히 tau 단백질 뿐 아니라, tau-결합단백질의 이상과인산화 그리고 PHFs까지 관찰되는 등 거의 완벽한 치매증상을 보이는 뛰어난 효과가 있으므로, 치매 치료제 또는 조기 진단법의 개발 등에 유용하게 이용될 수 있다.As described above, in the present invention, a transgenic demented mouse was prepared by injecting a fusion gene combining the NSE / htau24 gene, which is a dementia-inducing gene, with the NSE promoter, into a mouse fertilized egg, and the demented mouse of the present invention is a NES / htau24 gene. Is an overdevelopment of dementia patients' behavioral abnormalities, and is particularly effective in treating dementia or early diagnosis, as it has an excellent effect of almost complete dementia symptoms such as abnormal phosphophosphorylation of tau-binding proteins and PHFs as well as tau proteins. It can be usefully used for the development of.
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5912410A (en) * | 1990-06-15 | 1999-06-15 | Scios Inc. | Transgenic non-human mice displaying the amyloid-forming pathology of alzheimer's disease |
WO2004007722A2 (en) | 2002-07-12 | 2004-01-22 | Axon Neuroscience Forschungs- Und Entwicklungs Gmbh | Transgenic animal expressing alzheimer’s tau protein |
KR20040110730A (en) | 2003-06-20 | 2004-12-31 | 식품의약품안전청 | Transgenic Alzheimer's mice expressing APPsw gene |
KR20050014993A (en) | 2003-08-01 | 2005-02-21 | 식품의약품안전청 | Double Transgenic Alzheimer's mice expressing mutant PS2 gene and APPsw gene |
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5912410A (en) * | 1990-06-15 | 1999-06-15 | Scios Inc. | Transgenic non-human mice displaying the amyloid-forming pathology of alzheimer's disease |
WO2004007722A2 (en) | 2002-07-12 | 2004-01-22 | Axon Neuroscience Forschungs- Und Entwicklungs Gmbh | Transgenic animal expressing alzheimer’s tau protein |
KR20040110730A (en) | 2003-06-20 | 2004-12-31 | 식품의약품안전청 | Transgenic Alzheimer's mice expressing APPsw gene |
KR20050014993A (en) | 2003-08-01 | 2005-02-21 | 식품의약품안전청 | Double Transgenic Alzheimer's mice expressing mutant PS2 gene and APPsw gene |
Non-Patent Citations (1)
Title |
---|
NCBI GenBank No.AB038993 |
Cited By (1)
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---|---|---|---|---|
KR101213325B1 (en) | 2010-02-18 | 2012-12-18 | 서울대학교산학협력단 | Transgenic dementia animal of Alzheimer type expressing Δtau gene and method for preparing the same |
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