小鼠pOct4-EGFP胚胎干细胞株的构建

Generation of pOct4-EGFP Mouse Embryonic Stem Clones

以建立p Oct4-EGFP转基因的小鼠胚胎干细胞(ESC)株,为研究ESC分化提供有力的示踪工具为目的,通过电穿孔法转染小鼠ESC,G418和荧光显微镜下挑选和观察所得阳性克隆,检测所得细胞胚胎干细胞特异性标志物AKP、Oct4及SSEA-1的表达,通过拟胚体(EB)诱导体外分化,RT-PCR检测三胚层的分化,通过Oct4免疫荧光观察p Oct4-EGFP ESC的Oct4与GFP的一致性表达,进一步通过诱导p Oct4-EGFP ESC分化观察GFP的表达情况,并用RT-PCR检测Oct4的表达.结果表明,形态学观察可见所得GFP阳性细胞呈克隆样鸟巢状生长,AKP染色呈强阳性,ESC特异性标志Oct4、SSEA-1表达阳性,RT-PCR结果显示p Oct4-EGFP ESC具有三胚层的分化能力.p Oct4-EGFP ESC免疫荧光及细胞分化结果均表明Oct4与GFP的表达一致.本研究成功建立了小鼠p Oct4-EGFP胚胎干细胞株,并可用于体外直观监测ESC分化的阶段性变化.

This paper is to establish a mouse embryonic stem cell（ ESC） line which contains the enhanced green fluorescence protein（ EGFP） reporter under the control of Oct4 promoter,and provide useful tool for studying the stages of differentiation of embryonic stem cells by direct observation of GFP expression. POct4- EGFP was transferred into mouse ESC by electroporation,and positive cells were selected by G418 scanning and fluorescence microscope observation. Expression of AKP,Oct- 4 and SSEA- 1 were detected for characterization of mouse p Oct4- EGFP ESC. Differentiation in vitro were induced by embryoid body（ EB） culture and RT- PCR were used for detecting the differentiation of three germ layers. Immunofluorescence staining of Oct4 on p Oct4- EGFP ESC was done for observing the GFP expression consistent with Oct4 expression. Differentiation in vitro were induced for further detecting consistency of the GFP expression with Oct4 expression which detected by RT- PCR. The results showed that the positive cells have Cloning growth,like bird＇s nest shape. Immunofluorescence staining demonstrated that these cells expressed the undifferentiated marker AKP,Oct4 and SSEA- 1. The result of RT- PCR demonstrated that p Oct4- EGFP ESC had the ability of the differentiation of three germ layers. The results of both immunofluorescence staining of Oct4 and differentiation in vitro proved that the GFP expression retained consistency with the Oct4 expression in mouse p Oct4- EGFP ESC. This study successfully established the mouse p Oct4- EGFP ESC which would exert beneficial influence on the visualized observation of the phase changes during the ESC differentiation in vitro.