过表达Gata6诱导分离猪胚外内胚层干细胞样细胞

摘要

本研究对Gata6 (G6)在猪诱导多能性干细胞(iPS细胞)以及胚外内胚层干细胞(XEN细胞)样细胞分离中的作用进行深入解析.从农大香猪的胎儿组织提取mRNAs,反转录为cDNAs,以此为模板克隆所需基因,构建逆转录病毒质粒pMXs-pOct4、pMXs-pSox2、pMXs-pKlf4、pMXs-pMyc、pMXs-pGata6、pMXs-pLin28、pMXs-pTbx3以及pMXs-pNanog,运用不同的基因组合感染猪胎儿成纤维细胞(PEF).结果表明,Gata6与Oct4、Sox2、Klf4和mMyc(简称G6OSKM)共转染可以提高早期重编程效率,Gata6代替Oct4,与Sox2、Klf4和mMyc(简称G6SKM)可以诱导猪胎儿成纤维细胞为iPS(Induced pluripotent stem)细胞,但与OSKM(Oct4、Sox2、Klf4和mMyc)组相比,重编程效率下降.Gata6的过表达使获得的猪iPS细胞在重编程后期发生了分化,形态类似XEN细胞,这些细胞在小鼠胚外内胚层干细胞培养液培养可以维持XEN细胞的形态,表达胚外内胚层干细胞的标记基因,如Gata4、Gata6和Sox17.OSKM 4因子获得的猪iPS细胞过表达Gata6后,也可以产生胚外内胚层干细胞样细胞.综上表明,过表达Gata6可以进行猪胚外内胚层干细胞样细胞的分离,为从正常胚胎对猪XEN细胞的分离与建系,以及猪早期胚胎调控机制的解析提供理论依据.%In this study,the role of Gata6 in porcine induced pluripotent stem cell (iPS cell) and extraembryonic endoderm stem cell (XEN cell) isolation was analyzed deeply.The required genes were cloned using templates of cDNAs,which were reverse transcripted from the mRNAs of the fetal tissues of Nongdaxiang pigs,and the retroviral plasmids pMXs-pOct4,pMXs-pSox2,pMXs-pKlf4,pMXs-pMyc,pMXs-pGata6,pMXs-pLin28,pMXs-pTbx3 and pMXs-pNanog were constructed,then,porcine fetal fibroblasts were infected with different combinations of genes.The results showed that co-transfection of Gata6 with Oct4,Sox2,Klf4 and mMyc (G6OSKM) could promote early reprogramming efficiency.Gata6 replacement of Oct4,with Sox2,Klf4 and mMyc (G6SKM),also could induce porcine fetal fibroblasts to iPS cells,but the reprogramming efficiency decreased,compared with the control group of OSKM (Oct4,Sox2,Klf4 and mMyc).Over expression of Gata6 forced pig iPS cells to undergo differentiation at the late stage of repro gramming,and changed into XEN cell like cells morphologically.These cells could maintain the morphology of XEN cells in mouse XEN cell culture medium,express XEN cell marker genes such as Gata4,Gata6 and Sox17.Overexpression of Gata6 in porcine iPS cells obtained by OSKM 4 factors also produced XEN cell like cells.The results indicate that overexpression of Gata6 can generate porcine XEN cell like cells,can provide theoretical basis for the isolation and derivation of porcine XEN cells from normal embryos and the exploration of the mechanisms of porcine early embryo developmental regulation.