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태아섬유아세포와 중간엽줄기세포를 이용한 체세포 복제돼지 생산 효율

Cloning Efficiency of Porcine Somatic Cell Nuclear Transfer Compared with Fetal Fibroblasts and Mesenchymal Stem Cells

박수현|조성근|김민주|김병우|주예은|조병욱|신택순|서자겸|허창기|김명후|심보웅|김효정
동물생명과학연구소 (Institute of Animal Life Science)|2022.03|동물자원연구 (ANNALS OF ANIMAL RESOURCE SCIENCES)|제33권 제1호|pp. 14-22|DOI 10.12718/AARS.2022.33.1.14

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Mesenchymal stem cellsSomatic cell nuclear transferFetal fibroblastsMini-pigDonor cell
주제분류축산학

초록

돼지에서 체세포 핵이식은 복제동물의 기초연구와 더불어, 유전자가 편집된 세포를 활용하여 유용 의료용 단백질의 생산, 이종장기이식 그리고 질환 모델 등 형질전환 돼지를 생산하는데 핵심적인 방법으로 사용되고 있다. 많은 연구자들에 의해 복제동물 생산 효율을 높이기 위한 연구가 진행되고 있으나, 아직도 돼지에서 체세포 핵이식을 통한 복제동물의 생산 효율이 낮은 실정이다. 체세포 핵이식에 있어 공여세포는 이러한 복제동물의 생산 효율에 영향을 미치는 매우 중요한 요소이므로 핵이식 효율 향상을 제고하기 위하여 본 연구에서는 중간엽줄기세포와 태아섬유아세포를 공여세포로 사용하여 복제수정란의 체외 발달률과 산자생산효율을 비교하였다. 먼저, 대퇴부와 태아로부터 각각 중간엽줄기세포와 태아섬유아세포를 분리한 후, 구축된 세포주를 이용하여 체세포 핵이식을 수행하여 이를 체외 배양하였으며, 복제 산자를 얻기 위하여 대리모에 이식하여 효율을 확인하였다. 핵이식수정란의 체외배양 2일째의 할구분할률은 중간엽줄기세포군이 태아섬유아세포군에 비하여 유의적으로 높은 결과를 나타내었다(81.5%와 72.0%) (p<0.05). 하지만 배반포배로의 발 달률(15.1%와 20.8%) 및 배반포의 할구(30.2±7.24와 34.3±6.46) 분석에서는 유의적 차이가 없는 결과를 확인하였다. 또한 대리모에 수정란이식을 통해 중간엽줄기세포 및 태아섬유아세포 군에서 각각 9마리와 19마리의 산자를 얻을 수 있었으며, 이에 관련된 임신율(50%와 52.3%), 출산율(40%와 45.4%)에서는 유사한 결과임을 확인하였다. 또한 수정란 이식 시 대리모 난소의 출혈체의 수가 임신, 유산, 분만율에 미치는 영향을 확인한 결과에서도 중간엽줄기세포 및 태아섬유아세포군에서 차이가 없음을 확인하였다. 이와 같은 결과로 중간엽줄기세포 및 섬유아세포는 체세포 핵이식을 통한 복제 수정란 및 산자 생산에 모두 유용한 공여세포임을 확인하였으며, 향후 복제 돼지 생산의 효율 증진을 위한 중요한 기초자료로 활용될 수 있을 것으로 기대된다.

Abstract

Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

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