中文摘要：

建立含有TetO-FUW-OSKM和FUW-M2rtTA的第二代猪（Sus scrofa domesticus）成纤维细胞,使其在添加强力霉素（doxycycline,DOX）而无需再次感染病毒的条件下可以重编程.将慢病毒（Lentiviral）质粒四环素调控基因（TetO）-FUW-OSKM和FUW-M2rtTA同时感染猪胎儿成纤维细胞（porcine embryonic fibroblasts,PEF）,在添加DOX的培养条件下,形成诱导多能干细胞（induced pluripotent stem cells,iPSCs）.随后,将iPSCs通过形成拟胚体（embryoid body,EB）再分化为成纤维样细胞,即TetO-PEF细胞.TetO-PEF携带TetO-FUW-OSKM和FUW-M2rtTA两个载体,且外源四因子拷贝数一致,在±DOX条件下调控四因子表达,直接驱动细胞重编程.本研究建立了TetO-PEF细胞系,为优化猪iPSCs培养条件提供了新的细胞资源.

英文摘要：

As porcine body size, physical structure, and metabolism are similar to that of humans, pig（Sus scrofa domesticus） is suitable to be human disease and regenerative medicine research model. Porcine induced pluripotent stem cells （iPSCs） have been generated by using a cocktail of defined transcription factors, however, the inserted gene copy is different. Here, we generated porcine secondary fibroblasts which contained tetracycline operator（TetO）-FUW-OSKM and FUW-M2rtTA and it could reprogram in the presence of the doxycycline（DOX） without further viral infection. The lentiviruses （TetO-FUW-OSKM and FUW- M2rtTA） infected porcine embryo fibroblasts（PEF） were reprogrammed into primary iPSCs in the iPSCs medium with DOX. The primary iPSCs were positive for alkaline phosphatase （AP） staining and only could be passaged with leukemia inhibitory factor（LIF） and basic fibroblast growth factor（bFGF） on the Matrigel. The fibroblasts were differentiated by embryoid body formation and termed TetO-PEF. The TetO-PEF contains TetO-FUW-OSKM and FUW-M2rtTA which the inserted gene copy was the same. Without further viral infection Could the TetO-PEF be reprogramming in the iPSCs medium with DOX. The iPSCs obtained in this way were termed the secondary iPSCs. Reprogramming of iPSCs from the TetO-PEF was more efficient and faster than that from fibroblasts. The secondary iPSCs were characterized by AP staining and with expression of OCT4, SOX2,SSEA1,SSEA4 and TRA- 1-60 and without expression of TRA- 1-81. A system was set up for reprogramming of porcine somatic cells to pluripotency only with DOX, which will provide cell for the optimization of porcine iPSCs culture medium.