KR101174493B1 - Transgenic pig with overexpression of mouse PEA15, and method for preparing thereof - Google Patents
Transgenic pig with overexpression of mouse PEA15, and method for preparing thereof Download PDFInfo
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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
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases
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Abstract
본 발명은 제2형 당뇨병 환자에게서 관찰되는 PEA15 유전자의 과발현 현상을 생쥐 PEA15 유전자를 사용하여 돼지에서 과발현 되도록 한 것으로, 당뇨병 치료제 개발 및 효능검증과 병인해석 등에 필요한 전임상 당뇨질환 모델동물의 개발 이전 단계의 것이다. The present invention is to overexpress the phenomenon of PEA15 gene observed in type 2 diabetes patients in pigs using the mouse PEA15 gene, the pre-development of the development of preclinical diabetic disease model animals needed for the development and treatment of diabetes mellitus and etiology analysis Will.
Description
본 발명은 제2형 당뇨병 환자에게서 관찰되는 PEA15 유전자의 과발현 현상을 생쥐 PEA15 유전자를 사용하여 돼지에서 과발현 되도록 한 것으로, 당뇨병 치료제 개발 및 효능검증과 병인해석 등에 필요한 전임상 당뇨질환 모델동물의 개발 이전 단계의 것이다. The present invention is to overexpress the phenomenon of PEA15 gene observed in
인슐린 의존성인 제1형 당뇨병은 모든 당뇨병의 경우 10%에 해당하는 반면, 인슐린 비의존성인 제2형 당뇨병은 90%에 달한다. 제1형 당뇨병은 주로 소아기 또는 사춘기의 정상체중 혹은 야윈 사람에게서 발병되는 반면, 제2형 당뇨병은 40세 이후 과체중인 사람에게서 두드러지게 발병되나 최근에는 젊은 환자들도 증가하는 추세이다(Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2003). 이러한 증가 추세는 2025년 당뇨병 환자는 약 680만 명으로 늘어날 것으로 추산되며, 이와 함께 당뇨병 치료제 특허출원 건수도 증가하고 있다.Insulin-
한편, 동물의 형질전환 기술의 발전은 생명공학의 전반적인 분야에 영향을 미치고 있으며 인간의 각종 질환에 대한 새로운 치료제의 개발 및 효능 검증과 질병의 발병원인 해석 등을 위한 질환모델 동물의 생산에도 응용이 되고 있다. 특히, 당뇨병 관련 질환모델 동물은 설치류를 중심으로 개발되어 왔지만, 인간의 질병에 대해 전반적으로 이해하고 반영하기에는 다소 한계가 있었다. 가령, 마우스는 기초 유전적 메카니즘을 제외하고는 인간에 대해서 상대적으로 빈약한 모델이다(Petters & Sommer, 2000). 그래서 인간과 생리학적으로 유사한 중대동물의 질환모델 동물(특히 돼지)의 필요성이 대두되고 있다. 돼지는 해부학적 및 생리학적으로 인간과 매우 유사하며 새로운 치료제 및 치료법의 개발에 유용하게 이용될 것이다(Larsen과 Rolin, 2004; Lunney, 2007).Meanwhile, the development of animal transformation technology affects the overall field of biotechnology, and is also applied to the production of disease model animals for the development and validation of new therapeutic agents for human diseases and the interpretation of the causes of diseases. It is becoming. In particular, diabetes-related disease model animals have been developed around rodents, but there are some limitations in understanding and reflecting human disease in general. For example, mice are relatively poor models for humans with the exception of the underlying genetic mechanisms (Petters & Sommer, 2000). Therefore, the necessity of disease model animals (especially pigs) of large animals that are physiologically similar to humans is emerging. Pigs are anatomically and physiologically very similar to humans and will be useful for the development of new therapies and therapies (Larsen and Rolin, 2004; Lunney, 2007).
1998년 Condorelli 등은 제2형 당뇨병 환자에게서 PEA15(phosphoprotein enriched in astrocytes 15) 또는 PED(phosphoprotein enriched in diabetes)유전자가 과발현되는 사실을 발견하였고, 이후 이 유전자의 과발현이 인슐린 신호에 의해 세포막으로 이동해서 당을 세포 내로 유입해야 할 당 수송체인 GLUT4(glucose transport 4)의 막 이동을 저해시켜 세포내로 당 유입을 감소시킨다고 보고하였다(Vigliotta 등, 2004). PEA15는 다양한 조직에서 광범위하게 발현되고 포유류 사이에서 고도로 잘 보존되어 있는 15kDa의 세포질 단백질이다(Danziger 등, 1995; Estelles 등, 1996; Ramos 등, 2000). PEA15 단백질은 N-말단에 단백질-단백질 상호작용 영역인 Death Effector Domain(DED) 및 Nuclear Export Sequence(NES) 영역과 C-말단에 Ser104 및 Ser116의 인산화 부위를 가지며, 각각 Protein kinase C(PKC) 및 Calcium-calmodulin Kinase Ⅱ(CaMKⅡ)에 의해 인산화된다(Kubes 등, 1998; Trencia 등, 2003). 또한, 이 단백질은 다양한 메커니즘을 통하여 몇몇의 세포사멸 경로를 저해하고, 종양 발달 및 antineoplastic제에 대한 감수성에 있어 중요한 역할을 한다.In 1998, Condorelli et al. Discovered that overexpression of either PEA15 (phosphoprotein enriched in astrocytes 15) or PED (phosphoprotein enriched in diabetes) genes in patients with
지금까지 돼지를 이용한 당뇨 질환모델들은 대부분 streptozotosin이나 alloxan 등에 의한 외과적 처리로 췌장 세포를 파괴하는 방법에 의해 당뇨병을 유도하거나, 혹은 선택적인 육종 방법 등에 의해 당뇨병의 표현형을 도출했다(Junod 등, 1969; Lenzen과 Paten, 1988). 최근에 형질전환 기술을 이용한 당뇨질환 모델 돼지의 생산에 있어 일본의 메이지대학 Nagashima 교수팀은 당뇨병 모델 형질전환 돼지를 간세포핵인자를 이용하여 개발하였으며, 독일의 Ludwig-Maximilians대학의 Kessler 팀에서는 인크레틴 호르몬 수용체를 이용한 동물모델 시스템으로 제2형 당뇨병에 대한 병인해석에 응용할 모델동물을 개발하였다. 국내에선 2009년 8월 황우석 박사팀이 당뇨질환 모델돼지를 개발하겠다고 발표한 것이 유일한 상황이다.Until now, most diabetic disease models using pigs induced diabetes by the method of destroying pancreatic cells by surgical treatment with streptozotosin or alloxan, or derived the phenotype of diabetes by selective breeding methods (Junod et al., 1969). Lenzen and Paten, 1988). Recently, a team of researchers from Meiji University, Nagashima, Japan, developed a diabetic model pig using transgenic technology using a hepatocyte nuclear factor, and the Kessler team from Ludwig-Maximilians University in Germany, Incredin. An animal model system using hormone receptors has been developed to apply to the pathogenesis of
새로운 신약이 개발되기까지는 엄청난 연구비와 시간이 투자되어야 한다. 특히 임상시험단계에서의 긍정적인 결과를 도출하기 위해선 전 임상시험단계는 무엇보다 중요하다. 일반적으로 전 임상시험은 동물실험을 통해서 독성, 약리, 체내동태 등을 파악하여 인간에게 적용할 초기 투여용량, 부작용의 spectrum, 독성용량 등을 예측한다. 하지만, 이들 실험동물 특히 마우스는 그 다양성이 부족(예: 근교계 마우스, 즉 한품종)하고 인간과 유전적으로 비슷하지만 같지는 않다. 이러한 사실들은 실험동물의 한계성을 나타낸다. 반면, 돼지는 생리학, 병리생리학 및 이들의 반응에 대해서 인간과 비슷한 점이 많다. A huge amount of research and time must be invested in the development of new drugs. In particular, in order to derive positive results in the clinical trial phase, the entire clinical trial phase is of paramount importance. In general, preclinical trials identify toxicity, pharmacology, and body dynamics through animal experiments to predict initial dosage, spectrum of adverse effects, and toxic doses to be applied to humans. However, these laboratory animals, in particular mice, lack a variety (eg, inbred mice), which are genetically similar but not identical to humans. These facts indicate the limitations of experimental animals. Pigs, on the other hand, have many similarities to humans in physiology, pathophysiology, and their responses.
이에 본 발명자는 이러한 돼지의 장점을 기초로 하여 당뇨질환 전임상 모델동물의 개발에 앞서, 당뇨병 유발 예상유전자인 PEA15가 과발현되는 형질전환 동물을 제공하는 것이다. 이로써 본 유전자와 당뇨병과의 관계 및 기능해석, 당뇨질환 모델동물로서의 가능성 등 관련 연구 분야와 기초연구에 필요한 형질전환 동물을 공급하여 최종적으로는 제2형 당뇨질환 모델동물의 개발에 기여하고자 한다.Therefore, the present inventors provide a transgenic animal overexpressing the antidiabetic gene PEA15 prior to the development of a preclinical model animal with diabetes disease based on the advantages of such a pig. The aim is to contribute to the development of
본 발명의 일 측면은 생쥐 PEA15 유전자를 과발현시키는 벡터 pCAGGS-mPEA15(KACC 95103P)에 의해 형질전환된 생쥐 PEA15 유전자가 과발현된 형질전환 돼지를 제공한다.One aspect of the present invention provides a transgenic pig overexpressing the mouse PEA15 gene transformed with the vector pCAGGS-mPEA15 (KACC 95103P) overexpressing the mouse PEA15 gene.
상기 생쥐 PEA15 유전자가 과발현된 형질전환 돼지는 서열번호 2 및 서열번호 3으로 이루어진 프라이머 쌍을 이용하여 RT-PCR RFLP법으로 검정할 수 있다. The transgenic pigs overexpressed with the mouse PEA15 gene may be assayed by RT-PCR RFLP method using a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
또한, 상기 RT-PCR RFLP법은 상기 서열번호 2 및 서열번호 3으로 이루어진 프라이머 쌍을 이용한 RT-PCR을 통하여 생산된 산물을 제한효소로 절단하여 생성된 DNA 단편의 길이를 판독하여 형질전환 돼지를 구별할 수 있다. In addition, the RT-PCR RFLP method is a transgenic pig by reading the length of the DNA fragment produced by cutting the product produced by RT-PCR using a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3 with a restriction enzyme Can be distinguished.
또한, 상기 제한효소는 EcoR V 제한효소일 수 있다.
In addition, the restriction enzyme may be an EcoR V restriction enzyme.
본 발명의 다른 측면은 생쥐 PEA15 유전자를 과발현시키는 벡터 pCAGGS-mPEA15(KACC 95103P)에 의해 형질전환된 생쥐 PEA15 유전자가 과발현된 형질전환 돼지 수정란을 제공한다.
Another aspect of the present invention provides a transgenic pig fertilized egg overexpressed with the mouse PEA15 gene transformed with the vector pCAGGS-mPEA15 (KACC 95103P) overexpressing the mouse PEA15 gene.
본 발명의 또 다른 측면은 생쥐 PEA15 유전자를 과발현시키는 벡터 pCAGGS-mPEA15(KACC 95103P)를 이용하여 형질전환시키는 것을 포함하는 생쥐 PEA15 유전자가 과발현된 형질전환 돼지의 생산방법을 제공한다.
Another aspect of the present invention provides a method of producing a transgenic pig overexpressing a mouse PEA15 gene, which comprises transforming with a vector pCAGGS-mPEA15 (KACC 95103P) that overexpresses the mouse PEA15 gene.
상기와 같은 본 발명의 목적을 달성하기 위해서, 국제공동연구 협약을 통하여 일본 Tokyo대학교 의과학연구소 Nakauchi 교수로부터 제공받은 생쥐 PEA15 유전자를 과발현시키는 벡터 pCAGGS-mPEA15(도 1 참조)를 이용하며, 그 하기의 염기서열은 서열번호 1로 표현된다. 상기 벡터 pCAGGS-mPEA15는 2010년 3월 18일에 국립농업과학원 농업유전자원센터에 기탁하였다(수탁번호 KACC 95103P).In order to achieve the object of the present invention as described above, using the vector pCAGGS-mPEA15 (see Fig. 1) overexpressing the mouse PEA15 gene provided by Professor Nakauchi of the Institute of Medical Sciences, University of Tokyo, Japan, through the International Joint Research Agreement. The nucleotide sequence is represented by SEQ ID NO: 1. The vector pCAGGS-mPEA15 was deposited on March 18, 2010 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center (Accession Number KACC 95103P).
또한 본 발명에서는 목적유전자의 염색체(chromosome) 내로의 삽입 여부, 특히 내인성 유전자(endogenouse gene)와 외인성 유전자(exogenouse gene)의 구별을 통한 형질전환 개체의 판별법인 RT-PCR RFLP법을 통해 형질전환 돼지를 검정한다. 이 경우 내인성 유전자는 돼지 본래의 PEA15 유전자를 말하며, 외인성 유전자는 외래유전자 즉, 생쥐 PEA15 유전자를 말한다.In addition, in the present invention, transgenic pigs are determined through RT-PCR RFLP, which is a method for discriminating a transgenic individual by distinguishing between the endogenous gene and the exogenous gene of an endogenous gene. Test. In this case, the endogenous gene refers to the pig native PEA15 gene, and the exogenous gene refers to the foreign gene, that is, the mouse PEA15 gene.
상기의 분석법에 사용되는 RT-PCR 프라이머 염기서열은 다음과 같다. 외인성 및 내인성 유전자의 공통되는 부분에서 프라이머 염기서열을 설계하는 것을 특징으로 한다(도 2).The RT-PCR primer base sequence used in the above assay is as follows. It is characterized by designing primer sequences in common parts of exogenous and endogenous genes (FIG. 2).
서열번호 2 : Forward primer: 5'-GGACATCCCCAGTGAGAAGA-3'SEQ ID NO: Forward primer: 5'-GGACATCCCCAGTGAGAAGA-3 '
서열번호 3 : Reverse primer: 5'-TACGGGTTAGCTTGGTGTCC-3'
SEQ ID NO: 3 Reverse primer: 5'-TACGGGTTAGCTTGGTGTCC-3 '
또한 플라스미드 발현벡터를 제한효소 Pvu I으로 처리하여 플라스미드 벡터를 직선화시켜 1세포기 수정란에 유전자를 미세주입하여 대리모를 통하여 생산된 생쥐 PEA15 유전자가 과발현되는 형질전환 돼지 및 그 후대를 제공한다.In addition, the plasmid expression vector was treated with restriction enzyme Pvu I to linearize the plasmid vector to microinject the gene into the 1-cell fertilized egg, thereby providing a transgenic pig and its later overexpressed mouse PEA15 gene produced through surrogate mothers.
본 발명을 통하여 생쥐 PEA15 유전자가 과발현되는 형질전환돼지에 있어 본 유전자의 과발현과 당뇨병 발병의 병인해석에 활용할 수 있고, 또한 개발된 개체가 병리학적으로 당뇨병이 유발될 시 당뇨병 치료제 개발과 효능 검증 및 관련 연구 분야에 활용 될 것으로 기대된다. 또한 본 형질전환 개체의 수정란을 이용하여 새로운 당뇨질환 유전자를 삽입시켜 질환발병의 고효율성을 높이는데 활용이 가능하다.The present invention can be utilized for the overexpression of this gene and the pathogenesis of diabetes onset in transgenic pigs overexpressing the mouse PEA15 gene. It is expected to be utilized in related research fields. In addition, by inserting a new diabetic disease gene using the fertilized egg of the transgenic individual it can be utilized to increase the efficiency of disease development.
도 1은 생쥐 PEA15(mPEA15) 과발현 벡터pCAGGS-mPEA15의 구조를 나타내는 개열지도이다.
도 2는 마우스 및 돼지의 PEA15 염기서열을 비교하고 또한 프라이머와 제한효소의 위치를 표시한 것이다.
도 3은 형질전환 개체의 검정을 위한 RT-PCR RFLP 분석법의 모식도 및 전기영동 사진이다.
도 4는 생쥐 PEA15 유전자가 미세주입된 돼지 수정란으로부터 태어난 새끼(F0)를 RT-PCR RFLP 분석한 전기영동 사진이다.
도 5는 생쥐PEA15 형질전환 돼지 근육조직에서의 mRNA 및 단백질의 과발현을 나타낸 것이다.
도 6은 형질전환된 돼지에서 GFP 형광유전자의 발현 여부를 관찰한 결과이다.1 is a cleavage map showing the structure of mouse PEA15 (mPEA15) overexpression vector pCAGGS-mPEA15.
Figure 2 compares the PEA15 sequences of mice and pigs and also shows the positions of the primers and restriction enzymes.
Figure 3 is a schematic diagram and electrophoresis picture of the RT-PCR RFLP assay for the assay of transgenic individuals.
Figure 4 is an electrophoresis picture of the RT-PCR RFLP analysis of pups (F0) born from pig fertilized eggs microinjected with the mouse PEA15 gene.
Figure 5 shows overexpression of mRNA and protein in mouse PEA15 transgenic porcine muscle tissue.
Figure 6 shows the results of observing the expression of GFP fluorescence gene in transformed pigs.
이하, 구체적인 실시예를 통하여 본 발명을 더 상세히 설명하고자 한다.
Hereinafter, the present invention will be described in more detail with reference to specific examples.
[실시예 1] 생쥐 PEA15 발현벡터의 특징 및 미세주입용 유전자 제작 Example 1 Characterization of Mouse PEA15 Expression Vector and Preparation of Microinjection Gene
도 1에 도시된 8559bp(서열번호 1)의 pCAGGS-mPEA15 발현벡터는 CMV 프로모터-mPEA15-IRES-EGFP로 2개의 유전자를 발현시키는 구조를 가진다. 생쥐 PEA15 유전자를 과발현시키는 벡터 pCAGGS-mPEA15는 국제공동연구 협약을 통하여 일본 Tokyo대학교 의과학연구소 Nakauchi 교수로부터 제공받았다. 이 발현벡터를 Pvu I 제한효소로 절단하여 직선화시킨 후 0.9% 아가로스 겔에 전기영동하였고, 이들의 DNA 단편을 아가로스 겔로부터 회수하여 정제 후, 최종농도 2~4ng/㎕로 희석하여 유전자 미세주입 시 사용하였다.
The pCAGGS-mPEA15 expression vector of 8559 bp (SEQ ID NO: 1) shown in FIG. 1 has a structure for expressing two genes with CMV promoter-mPEA15-IRES-EGFP. The vector pCAGGS-mPEA15, which overexpresses the mouse PEA15 gene, was provided by Professor Nakauchi of the Institute of Medical Science, University of Tokyo, Japan, through an international joint research agreement. The expression vector was digested with Pvu I restriction enzyme and linearized, followed by electrophoresis on a 0.9% agarose gel. The DNA fragments were recovered from the agarose gel, purified, and diluted to a final concentration of 2 to 4 ng / μl. Used at the time of injection.
[[ 실시예Example 2] 수정란 채란 및 이식과 유전자 미세주입 2] Embryo Transfer and Transplantation and Gene Microinjection
외경이 1㎜이고 길이가 13㎝인 유리관(Garner glass, USA)으로 고정용과 주입용의 micropipets을 제작하였다. 생쥐 PEA15 유전자를 포함하는 벡터(도 1 참조)인 외래유전자의 미세주입은 미세조작기(micromanipulator: Nikon, Japan)를 이용하여 1세포기 수정란에 미세주입하였다. 한편, 유전자를 주입할 수 있는 1세포기 단계의 수정란을 생산하고 그 회수율을 높이기 위해 200~230일령의 암컷 랜드레이스(Landrace)에 Altrenogest를 발정주기 12일부터 14일 사이에 매일 두당 사료 2㎏에 20㎎을 혼합하여 9일간 급여하였다. 그 후 과배란에 PMSG(Intervet International BV, Netherlands) 1,000IU를 근육주사한 후 72시간이 경과했을 때 750IU의 hCG를 근육 주사하여 과배란을 유기하였다. hCG(Intervet International BV, Netherlands) 주입 후 약 24시간 발정관찰한 후 발정이 온 개체는 2회에 걸쳐 자연종부를 시키고, 약 53시간 경과 후 외과적 방법에 의해 난관을 D-PBS로 관류하여 수정란을 채란하였다. 관류된 액체는 멸균시험관에 수집하여 실온에서 난자를 회수하여 검란을 실시하며 1세포기 난자를 이용하였다. 호르몬 처리에 의해서 발정이 동기화된 수란돈 또는 자연적으로 발정주기가 동기화된 수란돈에 대하여 전신마취를 한 후, 마취된 수란돈의 정중선을 중심으로 광범위하게 면도기를 이용하여 체모를 제거하고 소독을 실시 한 다음 정중선을 약 10~15㎝를 절개하여 자궁 및 난소를 노출시켰다. 난소표면의 황체의 존재를 확인한 후, 수정란 26개가 함유된 배양액의 채워진 polycatether(Cook, Austraila)를 난관 팽대부에 넣고, 수정란이 함유된 배양액을 주입하여 이식하였다. 이식이 완료된 자궁 및 난소를 복강내로 밀어 넣은 다음, sodium chondroitin sulfate 1%가 함유된 생리식염수로 생식기를 세척하여 장간흡착을 방지하였다. 마지막으로 절개부위를 봉합한 뒤 항생제를 투여한 후, 보온이 잘되는 돈방에서 마취가 풀릴 때까지 보호하였다.
The micropipets for fixation and injection were prepared from Garner glass, USA, having an outer diameter of 1 mm and a length of 13 cm. Microinjection of the foreign gene, a vector containing the mouse PEA15 gene (see FIG. 1), was microinjected into a one-celled fertilized egg using a micromanipulator (Nikon, Japan). Meanwhile, Altrenogest was injected into female Landrace (200-230-day-old female) Landrace (Landrace) in order to produce 1-cell stage embryos capable of injecting genes, and 2kg of feed per head daily between 12 and 14 days of estrous cycle. 20 mg was mixed and fed for 9 days. After 72 hours of intramuscular injection of 1,000 IU of PMSG (Intervet International BV, Netherlands) in over ovulation, 750 IU of hCG was intramuscularly injected to induce hyperovulation. After estrus observation about 24 hours after injection of hCG (Intervet International BV, Netherlands), the estroused subjects had two spontaneous swellings, and after about 53 hours, the oviduct was perfused to D-PBS by surgical methods. Was filled. The perfused liquid was collected in a sterile test tube to recover the eggs at room temperature to perform egg examination and use a one-celled egg. After general anesthesia for estranged synchronized hormoned or naturally estranged hormonal by hormonal treatment, hair removal and disinfection are carried out using a razor extensively around the midline of the anesthetized lanthanide The midline was then incised approximately 10-15 cm to expose the uterus and ovaries. After confirming the presence of the corpus luteum on the surface of the ovary, the filled polycatether (Cook, Austraila) of the culture medium containing 26 fertilized eggs was placed in the tubal bulge, and the culture medium containing the fertilized egg was injected. After transplantation, the uterus and ovary were inserted into the abdominal cavity, and the genitals were washed with saline containing 1% sodium chondroitin sulfate to prevent intestinal absorption. Finally, the incision was closed and antibiotics were administered, and then protected until the anesthesia was released in a well-heated pig room.
[[ 실시예Example 3] 3] RTRT -- PCRPCR RFLPRFLP 법에 의한 형질전환 개체의 검정Assay of Transgenic Individuals by the Law
대리모로부터 태어난 자돈의 혈액 및 꼬리조직에서 total RNA를 ZR Whole-Blood Total RNA Kit(Zymo Research, USA) 및 TRIzol reagent(Invitrogen, USA)를 이용하여 제공된 방법에 의해 각각 추출하였다. cDNA는 1㎍의 total RNA를 1st Strand cDNA Synthesis Kit(Roche, USA)의 사용법에 따라 최종 20㎕에 반응시켜 합성했다. RT-PCR은 TaKaRa PCR Thermal Cycler(TaKaRa, Japan)을 사용하여, 최초 94℃에서 5분간 반응시킨 뒤 94℃에서 30초, 60℃에서 30초, 72℃에서 30초를 총 35회 반복하여 증폭하였으며, 최종 72℃에서 5분간 반응시켰다. PEA15 유전자 증폭에는 Forward primer인 5'-GGACATCCCCAGTGAGAAGA-3'(서열번호 2)와 Reverse primer인 5'-TACGGGTTAGCTTGGTGTCC-3'(서열번호 3)를 이용하여 최종 50㎕에 반응시켜 318bp의 증폭산물을 생산했다. 증폭된 PCR 산물을 정제한 후, 증폭된 산물이 서로 다른 EcoR V 제한효소 위치를 가진다는 사실을 이용하여 EcoR V 제한효소로 절단하였다. 절단 후, 1.8% 아가로스 겔에 전기영동하여 외인성 유전자인 생쥐 PEA15와 내인성 유전자인 돼지 PEA15를 DNA 단편의 크기에 따라 구별하였으며, 그 모식도와 전기영동 결과를 도 3에 표시하였다.
Total RNA from pigs' blood and tail tissues born from surrogate mothers was extracted by the method provided using ZR Whole-Blood Total RNA Kit (Zymo Research, USA) and TRIzol reagent (Invitrogen, USA), respectively. cDNA was synthesized by reacting 1 μg total RNA with the final 20 μl according to the usage of 1st Strand cDNA Synthesis Kit (Roche, USA). RT-PCR was amplified by TaKaRa PCR Thermal Cycler (TaKaRa, Japan) for 35 minutes at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds at 94 ° C for 5 minutes. The reaction was carried out at 72 ° C. for 5 minutes. For PEA15 gene amplification, 318bp amplified product was produced by reacting the final 50µl with 5'-GGACATCCCCAGTGAGAAGA-3 '(SEQ ID NO: 2) and reverse primer 5'-TACGGGTTAGCTTGGTGTCC-3' (SEQ ID NO: 3). did. The amplified PCR products were purified and then digested with EcoR V restriction enzymes using the fact that the amplified products had different EcoR V restriction enzyme positions. After cleavage, electrophoresis was performed on 1.8% agarose gel to distinguish the exogenous gene, mouse PEA15 and the endogenous gene, pig PEA15, according to the size of the DNA fragment, and the schematic and electrophoresis results are shown in FIG. 3.
[[ 실시예Example 4] 생쥐 4] mice PEA15PEA15 의 과발현 및 Overexpression of and GFPGFP 형광유전자의 발현분석 Expression Analysis of Fluorescent Genes
생쥐 PEA15 유전자가 삽입된 개체에 있어 본 유전자의 과발현 유무를 분석하기 위해서 근육조직에서 total RNA 및 단백질을 추출하여 이하와 같이 실시하였다. mRNA 발현량은 LightCycler FastStart DNA Master SYBR Green I(Roche, USA)를 이용하여 LightCycler(Roche, USA)로 분석했다. 최초 94℃에서 5분간 반응시킨 뒤 94℃에서 15초, 60℃에서 15초, 72℃에서 15초를 총 30회 반복하여 증폭하였으며, 최종 72℃에서 5분간 더 반응시켰다. PCR 반응 종결 후 melting curve 작성을 통하여 유전자 증폭의 정확성을 재확인하였다. 유전자 발현량의 내부 보정을 위하여 house keeping 유전자인 돼지의 GAPDH(glyceraldehyde-3-phosphate dehydrogenase)를 이용하였고, 유전자 발현량은 2-△Ct 방법을 통하여 분석하였다. 여기서 Ct값은 반응을 일으키는 최소 역가(threshold)의 cycle 수를 의미하며, △Ct 값은 PEA15 유전자의 Ct 값에서 GAPDH 유전자의 Ct값을 뺀 값을 의미한다(Livak와 Schmittgen, 2001). PEA15의 증폭 primer는 상기 Forward primer인 5'-GGACATCCCCAGTGAGAAGA-3'(서열번호 2)와 Reverse primer인 5'-TACGGGTTAGCTTGGTGTCC-3'(서열번호 3)를 이용하였고, GAPDH는 forward primer인 5'-ATTGCCCTCAACGACCACT-3'(서열번호 4)과 reverse primer인 5'-GGCCTCTCTCCTCCTCGC-3'(서열번호 5)을 이용하여 194bp의 PCR 증폭산물을 증폭시켰다. western blotting을 실시하여 단백질 발현량을 확인하였다. -80℃에서 보관 중인 조직을 protease inhibitor cocktail(Roche, USA)이 첨가된 M-PER 단백질 추출 용액(Thermo Scientific, USA)에서 homogenizer를 이용 분쇄하였다. 얼음에서 1시간 동안 방치하여 추가로 조직을 용해하였다. 원심분리기를 이용 조직 단백질만을 회수하여 분석 전까지 -80℃에서 보관하였다. 단백질의 농도는 Bradford assay 용액(Bio-RAD, USA)을 이용하여 결정하였다. 40㎍의 단백질을 SDS-PAGE 겔에서 분리시킨 후 PVDF membrane으로 전이시켰다. Western blot 분석을 위해 blocking buffer는 5% non-fat dried milk와 0.05% Tween-20이 첨가된 PBS 용액을 사용하였다. 1차 항체는 anti-rabbit polycolonal PEA15(Cell Signaling Technology, USA)를 사용하였고, 2차 항체는 anti-rabbit IgG-HRP(Santa Cruz, USA)를 사용하였다. GFP 형광유전자의 발현은 귀 조직의 일부를 채취하여 형광현미경을 통하여 관찰하였고, 발굽에서의 GFP의 발현은 육안으로 관찰하였다.
In order to analyze the overexpression of the present gene in the mouse PEA15 gene inserted subjects, total RNA and protein were extracted from muscle tissue and carried out as follows. mRNA expression was analyzed by LightCycler (Roche, USA) using LightCycler FastStart DNA Master SYBR Green I (Roche, USA). After the first reaction at 94 ° C. for 5 minutes, amplification was repeated a total of 30 times for 15 seconds at 94 ° C., 15 seconds at 60 ° C., and 15 seconds at 72 ° C., followed by further 5 minutes at 72 ° C. After completion of the PCR reaction, the accuracy of gene amplification was reconfirmed by preparing the melting curve. For the internal correction of gene expression level, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) of pig, a house keeping gene, was used and gene expression level was analyzed by 2- △ Ct method. Here, Ct value means the minimum number of cycles to cause a reaction, and △ Ct value means the Ct value of the PEA15 gene minus the Ct value of the GAPDH gene (Livak and Schmittgen, 2001). The amplification primers of PEA15 were 5'-GGACATCCCCAGTGAGAAGA-3 '(SEQ ID NO: 2) and the reverse primer 5'-TACGGGTTAGCTTGGTGTCC-3' (SEQ ID NO: 3). PCR amplification products of 194bp were amplified using -3 '(SEQ ID NO: 4) and reverse primer 5'-GGCCTCTCTCCTCCTCGC-3' (SEQ ID NO: 5). Western blotting was performed to confirm the amount of protein expression. Tissues stored at −80 ° C. were ground using a homogenizer in M-PER protein extract solution (Thermo Scientific, USA) to which a protease inhibitor cocktail (Roche, USA) was added. The tissues were further dissolved by standing on ice for 1 hour. Only tissue proteins were collected using a centrifuge and stored at −80 ° C. until analysis. Protein concentration was determined using Bradford assay solution (Bio-RAD, USA). 40 μg of protein was isolated on SDS-PAGE gel and then transferred to PVDF membrane. For blocking blot analysis, 5% non-fat dried milk and 0.05% Tween-20 added PBS solution were used as blocking buffer. The primary antibody used anti-rabbit polycolonal PEA15 (Cell Signaling Technology, USA) and the secondary antibody used anti-rabbit IgG-HRP (Santa Cruz, USA). The expression of GFP fluorescence gene was observed by fluorescence microscopy of a part of ear tissue, and the expression of GFP in the hoof was visually observed.
[[ 실시예Example 5] 형질전환 돼지의 확인 5] Identification of Transgenic Pigs
생쥐 PEA15 유전자가 미세주입된 돼지 수정란으로부터 태어난 새끼(F0)들의 total RNA를 이용한 RT-PCR RFLP법에 의해 F0 17번 개체 한 마리를 생산하였다(도 4). 이 형질전환 개체는 정상돼지와의 교배를 통해서 F1 후대를 생산하였으며, 또한 생쥐 PEA15 유전자가 후대로 전이됨을 확인하였다(표 1).
One
division
Surrogate
Transition rate
Production method
생쥐 PEA15 유전자의 과발현 분석은 F1 후대의 근육조직에서 분석한 결과, mRNA 수준은 형질전환 돼지가 정상돼지에 비해 상대적으로 약 200배, 단백질 수준에서는 약 7배 정도 과발현됨을 확인하였다(도 5). 또한 2개의 유전자를 발현시키는 본 벡터시스템의 특징에 의해 GFP 형광유전자의 발현도 귀 조직 및 발굽에서 확인하였다(도 6). 이러한 결과들 즉, 근육조직에서의 PEA15 mRNA 및 단백질의 과발현은 앞서 서술한 바와 같이 당 수송체 GLUT 4의 막으로의 이동을 저해시켜 세포내 당의 유입을 감소시킬 수 있다는 가능성을 암시한다.As a result of overexpression analysis of mouse PEA15 gene in the muscle tissue of the posterior F1, it was confirmed that the mRNA level is overexpressed about 200-fold and about 7-fold at the protein level of the transgenic pig (Fig. 5). In addition, the expression of the GFP fluorescent gene was also confirmed in the ear tissue and the hoof by the characteristics of the present vector system expressing two genes (FIG. 6). These results, ie, overexpression of PEA15 mRNA and protein in muscle tissue, suggest the possibility of inhibiting the migration of
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (6)
상기 생쥐 PEA15 유전자가 과발현된 형질전환 돼지는 서열번호 2 및 서열번호 3으로 이루어진 프라이머 쌍을 이용하여 RT-PCR RFLP법으로 검정하는 생쥐 PEA15 유전자가 과발현된 전임상 당뇨질환 모델 형질전환 돼지.The method of claim 1,
The transgenic pig overexpressing the mouse PEA15 gene is a preclinical diabetic disease model transgenic pig overexpressing the mouse PEA15 gene assayed by RT-PCR RFLP using a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
상기 RT-PCR RFLP법은 상기 서열번호 2 및 서열번호 3으로 이루어진 프라이머 쌍을 이용한 RT-PCR을 통하여 생산된 산물을 제한효소로 절단하여 생성된 DNA 단편의 길이를 판독하여 형질전환 돼지를 구별하는 생쥐 PEA15 유전자가 과발현된 전임상 당뇨질환 모델 형질전환 돼지.The method of claim 2,
The RT-PCR RFLP method distinguishes transgenic pigs by reading the length of a DNA fragment generated by cutting a product produced by RT-PCR using a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3 with a restriction enzyme. Preclinical Diabetes Disease Model Transgenic Pigs Overexpressed with Mouse PEA15 Gene.
상기 제한효소는 EcoR V 제한효소인 생쥐 PEA15 유전자가 과발현된 전임상 당뇨질환 모델 형질전환 돼지.The method of claim 3,
The restriction enzyme is a preclinical diabetes disease model transgenic pig overexpressing the mouse PEA15 gene, which is an EcoR V restriction enzyme.
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