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| A Porcine(Sus scrofa domesticus) Somatic Cell Line of Tetracycline Operator(TetO)-inducible System for Reprogramming |
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Abstract 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.
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Received: 28 April 2014
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ZHU Xiao-Feng, XU Hou-Qiang, CHEN Wei, CHEN Ying-Lian, NI Kai, WU Xiao-Min, NI Meng-Meng, LU Xian-Jun. Cloning, Expression and Bioinformatics Analysis of IGF-1 and IGF-2 Genes in Congjiangxiang Pigs (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(8): 1382-1391. |
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