绵羊Oct4基因启动子的表达活性分析

Expression and activity analysis of sheep Oct-4 gene promoter

Oct4(POU5f1)是诱导重编程的关键性因子,为检测克隆获得的绵羊Oct4基因启动子的表达活性,本研究将前期工作中克隆获得的绵羊Oct4基因启动子与携带有报告基因绿色荧光蛋白(GFP)的p Ac GFP1-N1载体相连接,构建由Oct4启动子驱动的真核表达载体(SPOct4-p Ac GFP1-N1),并采用脂质体转染法,分别转染小鼠和绵羊成纤维细胞、小鼠胚胎干细胞和小鼠胚胎,以检测其表达活性。结果表明:Oct4启动子序列含有Sp1结合位点和CAAT框等功能区;转染后,小鼠胚胎干细胞和小鼠胚胎中检测到绿色荧光,并且在转染72 h后达到最大荧光强度,而在小鼠成纤维细胞和绵羊成纤维细胞中未能观察到GFP的表达。说明由克隆获得的Oct4基因启动子驱动的真核表达载体具有在多能干细胞中特异的表达活性。

Oct -4 is a key factor in induced reprogramming. To detect the expression and activity of cloned sheep Oct -4 gene promoter, the Oct -4 gene promoter that obtained by the previous work was linked with vector pAcGFP1 - N1 carrying a reporter gene green fluorescent pro- tein （GFP） to construct a eukaryotic expression vector SPOct4 - pAcGFP1 - N1 driven by the Oct -4 promoter. Mouse and sheep fibroblasts, mouse embryonic stem cells and mouse embryos were transfected with SPOor4 - pAcGFPI - N1 to detect its expression and activity using liposome - mediated gene transfer method, respectively. The results showed that a Spl binding site, CAAT box and other functional areas existed in the Oct -4 promoter sequence; green fluorescence could be detected in both mouse embryonic stem cells and mouse embryos after transfec- tion, and the fluorescence intensity reached the maximum at 72 h after transfection. However, the expression of GFP was not observed in mouse and sheep fibroblasts. The results indicate that the eukaryotic expression vector SPOct4 - pAcGFP1 - N1 driven by the cloned Oct -4 gene promoter had specific expression and activity in pluripotent stem cells.