猪诱导多能干细胞(iPS)嵌合胚胎的制备  

The Production of Pig Induced Pluripotene Stem Cell(iPSC) Chimeric Embryo

作　　者：高星[1] 马洋洋[1] 成德[1] 马晓玲[1] 王华岩[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,杨凌712100

出　　处：《农业生物技术学报》2012年第12期1349-1358,共10页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2009CB941002和No.2011CBA01002)

摘　　要：猪诱导多能干细胞（iPS）具有自我更新和多向分化潜能，是医学和细胞生物学研究理想的材料。为了探索猪iPS细胞的胚胎嵌合能力，本实验采用piggyBac转座子系统PB[Act-RFP]DS和Act-PBase载体，共转染猪iPS细胞，获得带有红色荧光标记的猪iPS细胞株PS24-R。并通过显微注射方法，将供体细胞PS24-R注入到4～8细胞期的胚胎腔隙，制备嵌合胚胎，待其继续发育至囊胚阶段，统计PS24-R细胞在胚胎中的嵌合情况。结果显示，通过转座子系统piggyBac标记的猪PS24-R细胞能够稳定表达红色荧光，以其作为供体细胞制备猪嵌合胚胎能够有效观察iPS细胞在胚胎中的嵌合情况。将1、5和10个猪PS24-R细胞和1个猪PS24．R细胞团分别注射到猪胚胎中构建嵌合胚胎，随着猪iPS细胞注射数量的增加，注射胚胎的囊胚率逐渐下降，但嵌合比例逐渐升高。同时与孤雌胚胎相比，嵌合胚胎中多能基因OCT4、SOX2和？CANOG表达显著提高。由此可见，采用piggyBac转座子标记的猪PS24-R细胞能够在猪早期胚胎发育阶段实现嵌合，为iPS细胞嵌合猪的生产提供了基础资料。Pig induced pluripotene stem ceils （iPSCs） are ideal materials for regenerative medicine and cell biology research because of their self-renewal and multilineage differentiation potential. In order to explore the embryo chimeric ability of pig iPSCs, the piggyBac transposon vectors, PB [Act-RFP] DS and Act-PBase, were used to cotransfect pig iPSCs. The PS24-R, a pig iPS cell line, was successfully obtained which expressed red fluorescent protein （RFP）. We have got chimeric embryos through mieroinjecting the PS24-R cells into the 4-8 cell parthenogenetic embryos in vitro. Then we collected the statistics ofblastocyst rate and chimeric rate as the chimeric embryos developed to the blastocyst stage. The result showed that the PS24-R cell using piggyBac vectors to transfect the iPS cells could stably express RFP. The pig iPS cells could be effectively observed in chimeric embryos. We also tried to microinject one, five, ten PS24-R cells and one PS24-R clone respectively to pig embryos to produce chimeric embryos. Along with increasing of the number of donor cells, the chimericrate of embryos gradually increased, whereas, blastocyst rate declined. Compared with the parthenogenetic embryos, the expression lever of the stem cell pluripotent factors, OCT4（octamer binding transcription factor 4）, SOX2（sry related HMG box-2） and NA NOG, had significant upregulated in chimeric embryos. In summary, the pig iPS marked by PiggyBac vector can achieve chimerism in the pig early embryonic development stage. This work lays a foundation for the production of pig iPS chimeric animals.

关 键 词：猪 诱导多功能干细胞(iPS) 嵌合胚胎 PIGGYBAC转座子 孤雌胚胎 

分 类 号：S828.3[农业科学—畜牧学]