骨髓间充质干细胞为核供体的克隆绵羊的生产与鉴定  

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

作　　者：郭园[1] 刘宗正[2] 刘乐[3,4] 孟凡华[3,4] 张焱如[3,4] 周欢敏[3,4] 刘春霞[3,4] GUO Yuan LIU Zong-zheng LIU Le MENG Fan-hua ZHANG Yan-ru ZHOU Huan-min LIU Chun-xia(College of Vocational and Technical , Inner Mongolia Agricultural University, Baotou 014109, China Qingdao Animal Husbandry and genterinary Research Institute, Qingdao 266000, China College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China The Inner Mongolia Autonomous Region Key Laboratory of Biological Manufacturing, Hohhot 010018, China)

机构地区：[1]内蒙古农业大学职业技术学院,包头014109 [2]青岛市畜牧兽医研究所,青岛266000 [3]内蒙古农业大学生命科学学院,呼和浩特010018 [4]内蒙古自治区生物制造重点实验室,呼和浩特010018

出　　处：《中国畜牧兽医》2016年第11期3011-3018,共8页China Animal Husbandry & Veterinary Medicine

基　　金：内蒙古自然基金重大项目(2012ZD03);内蒙古自治区自然科学基金(2016MS0315);内蒙古自治区高等学校科学研究项目(NJZY16066);内蒙古农业大学生命科学学院师生协同科技创新项目

摘　　要：本研究旨在采用骨髓间充质干细胞(BMSCs)为核供体进行体细胞核移植,以获得成体干细胞克隆绵羊。选择幼体绵羊BMSCs为核供体,构建重构胚进行克隆胚胎移植,克隆绵羊诞生后,选取IASG上公布的绵羊10个微卫星标记位点,PCR扩增克隆羊、供体细胞及代孕母羊微卫星DNA,利用Quantity One软件进行基因分型,计算供体细胞与克隆羊之间的父子关系相对机会(RCP)值。结果显示,以幼体绵羊BMSCs为核供体,与去核成熟卵母细胞进行核移植,构建重构胚进行电融合,其融合率达80.62%,重构胚发育至桑椹胚阶段,经手术植入20只代孕母羊,获得克隆绵羊5只,成活3只。克隆羊基因型与供体细胞一致,供体细胞与克隆羊之间的RCP≥99.999%,证明克隆羊来自幼体绵羊BMSCs。本试验最终成功获得了首例以BMSCs为核供体的克隆绵羊。In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5lambs were born and only 3were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.

关 键 词：骨髓间充质干细胞 体细胞核移植 克隆绵羊 

分 类 号：Q813[生物学—生物工程]