建立高效表达猪LIF的猪胚胎成纤维细胞系

Establishment of Cell Lines of Porcine Embryonic Fibroblasts Effectively Expressing Porcine Leukemia Inhibitory Factor

该文目的是建立高效表达重组猪白血病抑制因子(leukemia inhibitory factor,LIF)的猪胚胎成纤维细胞(porcine embryonic fibroblasts,PEF)系PEF-p LIF,为下一步辅助建立和培养猪nave胚胎干细胞奠定基础。以猪胚胎成纤维细胞的总RNA为模板,利用RT-PCR的方法扩增猪白血病抑制因子基因(LIF),将LIF c DNA连接到真核表达载体p CAGDNA3的启动子下游,构建LIF基因真核表达载体p CAGDNA3-p LIF;利用核转染的方法将p CAGDNA3-p LIF质粒转入PEF;对转染细胞进行G418筛选,得到稳定高效表达重组猪LIF的PEF-p LIF;利用RT-PCR、Western blot鉴定PEF-p LIF中LIF基因及LIF表达情况;使用PEF-p LIF细胞作为饲养层培养小鼠胚胎干细胞,通过对小鼠胚胎干细胞进行碱性磷酸酶染色及细胞免疫荧光染色,对PEF-p LIF维持干细胞干性功能进行初步验证。实验结果显示,成功构建了真核表达载体p CAGDNA3-p LIF;并将其成功转入PEF中,获得高效表达重组猪LIF的PEF-p LIF;以PEF-p LIF作为饲养层成功培养克隆形态正常的小鼠胚胎干细胞。该研究表明,稳定高效表达重组猪LIF蛋白的猪胚胎成纤维细胞系PEF-p LIF可作为饲养层维持小鼠胚胎干细胞的未分化状态,可为下一步建立及培养猪nave胚胎干细胞提供条件。

The purpose of this study is to establish the cell lines（PEF-p LIF） of porcine embryonic fibroblasts（PEF） effectively expressing recombinant porcine leukemia inhibitory factor（p LIF） in order to lay the foundation for establishing stable nave porcine embryonic stem cells（p ESCs）. The p LIF genes were obtained from the total RNA of porcine embryonic fibroblasts by polymerase chain reaction. The p LIF c DNA fragment was inserted into the eukaryotic expression vector p CAGDNA3 to generate the eukaryotic expression plasmid p CAGDNA3-p LIF. After nuclear transfection of the plasmid p CAGDNA3-p LIF into PEF, the cell lines that effectively express p LIF stably were screened by G418. The expression of the LIF gene and recombinant p LIF was detected by RT-PCR and Western blot. The functions of PEF-p LIF maintaining the characteristics of stem cells were investigated by mouse embryonic stem cells（m ESCs） culture, the alkaline phosphates expression detection and immunocytofluorescent. The results showed that the eukaryotic expression plasmid p CAGDNA3-p LIF was constructed successfully. The cell lines expressing effectively the recombinant p LIF（PEF-p LIF） were successfully established and m ESCs could maintain formal morphology with them as feeder cells. The PEF overexpressing the recombinant p LIF（PEF-p LIF） were successfully established. As feeder cells, they could keep mouse embryonic stem cells at undifferentiated state. The derived PEF-p LIF cell lines could be possibly helpful for establishing stable nave p ESCs in the future.