Home > Browse Issues > Vol.37 No.7
Establishment of Cell Lines of Porcine Embryonic Fibroblasts Effectively Expressing Porcine Leukemia Inhibitory Factor
Yang Ning#, Zhao Lihua#, Zhang Manling, Jin Yong, Hou Daorong, Wu Zhaoqiang, Chen Yuan, Li Rongfeng*
Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China
Abstract: The purpose of this study is to establish the cell lines (PEF-pLIF) of porcine embryonic fibroblasts (PEF) effectively expressing recombinant porcine leukemia inhibitory factor (pLIF) in order to lay the foundation for establishing stable naïve porcine embryonic stem cells (pESCs). The pLIF genes were obtained from the total RNA of porcine embryonic fibroblasts by polymerase chain reaction. The pLIF cDNA fragment was inserted into the eukaryotic expression vector pCAGDNA3 to generate the eukaryotic expression plasmid pCAGDNA3-pLIF. After nuclear transfection of the plasmid pCAGDNA3-pLIF into PEF， the cell lines that effectively express pLIF stably were screened by G418. The expression of the LIF gene and recombinant pLIF was detected by RT-PCR and Western blot. The functions of PEF-pLIF maintaining the characteristics of stem cells were investigated by mouse embryonic stem cells (mESCs) culture， the alkaline phosphates expression detection and immunocytofluorescent. The results showed that the eukaryotic expression plasmid pCAGDNA3-pLIF was constructed successfully. The cell lines expressing effectively the recombinant pLIF (PEF-pLIF) were successfully established and mESCs could maintain formal morphology with them as feeder cells. The PEF overexpressing the recombinant pLIF (PEF-pLIF) were successfully established. As feeder cells， they could keep mouse embryonic stem cells at undifferentiated state. The derived PEF-pLIF cell lines could be possibly helpful for establishing stable naïve pESCs in the future.
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