南京医科大学学报（自然科学版）2017，Vol.37Issue(8)：915-925,11.DOI:10.7655/NYDXBNS20170801
猪naive-like诱导性多能干细胞系的建立及红色荧光蛋白标记
Establishment of porcine naive-like induced pluripotent stem cell line and labelling with red fluorescent protein
摘要
Abstract
Objective:To establish porcine naive-like induced pluripotent stem (iPS) cell line followed by labelling with the red fluorescent protein via nucleofection.The labelled porcine naive-like iPS cells could be used for tracing their development and differentiation.Methods:Firstly,bama miniature pig embryonic fibroblasts (PEF) were nucleofected with TetO-FUW-OSKM (OSKM:mouse derived transcriptional factors OCT4,SOX2,KLF4 and c-MYC) and FUW-M2rtTA vectors.Then,these cells were incubated in the medium with both leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF),supplemented with doxycychne hyclate.The established iPS cell lines were identified for their pluripotency.Secondly,the iPS cells were nucleofected with pEFlalpha-DsRed-Express Vector which could express the red fluorescent protein,then the maintainence of pluripotent characters of these labeled cells were analyzed.Results:The established iPS showed domed morphology and could survive long-term single-cell passaging (30 passages).They were alkaline-phosphatase positive,maintained a normal karyotype,expressed various pluripotency markers and required LIF/STAT3 signaling pathway for self renewal.The iPS cells could form embryonic bodies(EB),which expressed markers of three germ layers after culture in LIF free medium.The iPS cells were transfected with red fluorescent protein vector successfullyand kept showing red fluorescence for 10 passages.The results of alkaline phosphatase staining and immunofluorescence staining suggested that the pluripotency of these cells had not been compromised after red fluorescent protein labelling.Conclusion:The red fluorescent protein labeled porcine naive-like iPS cell lines were established successfully.关键词
猪诱导性多能干细胞系/多能性/核转染/红色荧光蛋白Key words
porcine iPS/pluripotency/nucleofection/red fluorescent protein分类
生物科学引用本文复制引用
姜海滨,张曼玲,赵丽华,金永,陈袁,王俊政,陈俏羽,李荣凤..猪naive-like诱导性多能干细胞系的建立及红色荧光蛋白标记[J].南京医科大学学报（自然科学版）,2017,37(8):915-925,11.基金项目
国家自然科学基金(31371487) （31371487）
