Huang Yongye, Li Zhanjun, Wang Anfeng, Han Xiaolei, Song Yuning, Yuan Lin, Li Tianye, Wang Bing, Lai Liangxue, Ouyang Hongsheng, Pang Daxin
College of Life and Health Sciences Northeastern University Shenyang China; Jilin Provincial Key Laboratory of Animal Embryo Engineering College of Animal Sciences Jilin University Changchun China.
Jilin Provincial Key Laboratory of Animal Embryo Engineering College of Animal Sciences Jilin University Changchun China.
FEBS Open Bio. 2016 Mar 15;6(4):285-302. doi: 10.1002/2211-5463.12037. eCollection 2016 Apr.
Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell.
猪嵌合体在多能性、胚胎发生和发育研究中具有重要价值。从体细胞核移植胚胎中产生嵌合仔猪具有重要意义。在本研究中，使用两种表达荧光标记增强型绿色荧光蛋白（EGFP）和tdTomato的细胞系作为供体细胞来生产重构胚胎。通过在4细胞阶段将两个源自EGFP细胞的胚胎与两个源自tdTomato细胞的胚胎聚集来产生嵌合胚胎，当聚集胚胎发育成囊胚时进行胚胎移植。成功诞生了活体猪嵌合体，并通过其肤色、基因整合、微卫星位点组成和荧光蛋白表达观察到嵌合现象。嵌合仔猪主要由表达EGFP的细胞组成，这种现象可能是由于tdTomato基因启动子的高甲基化所致。此外，tdTomato转染膀胱癌细胞后，致瘤性相关基因的表达水平发生了改变。结果表明，通过聚集克隆胚胎可以产生嵌合猪，并且克隆胚胎在随后的嵌合发育中的发育能力可能受到其供体细胞生长特性的影响。
