人胚胎干细胞外源基因的瞬时表达效率

Transient expression efficiency of foreign gene in human embryonic stem cells

背景:人类胚胎干细胞的修饰和操作技术多种多样,它们在效率、可靠性及安全性方面各有不同。目的:对比观察两种化学转染试剂转染含不同启动子的增强型绿色荧光蛋白表达载体在人胚胎干细胞中的表达效率。方法:采用Fugene HD和lipofectamine2000分别转染无滋养层培养的H9细胞,荧光显微镜下观察阳性克隆,流式细胞术分析不同载体pCAG-EGFP、pEGFP-N3在H9细胞中的表达效率。结果与结论:Fugene HD转染的pCAG-EGFP、pEGFP-N3两种载体在H9细胞中的表达效率分别为(42.45±3.32)%、(25.95±1.91)%,差异有显著性意义(P<0.05)。lipofectamine2000转染的pCAG-EGFP、pEGFP-N3两种载体在H9细胞中的表达效率分别为(1.94±0.18)%、(1.49±0.33)%。采用Fugene HD转染的表达效率均高于lipofectamine2000转染的表达效率(P<0.05)。结果显示在人胚胎干细胞的H9细胞系中,Fugene HD的转染效率显著高于lipofectamine2000;CAG启动子驱动的增强型绿色荧光蛋白表达效率显著高于CMV启动子。

BACKGROUND：Technologies designed to allow modification and manipulation of human embryonic stem cells（hESCs） is numerous and various in the complexity of their efficiency,reliability and safety.OBJECTIVE：To compare effect of different enhanced green fluorescent protein（EGFP） expression vectors with different promoter on expression efficiency in human embryonic stem cells（hESCs）.METHODS：The H9 cells cultured with non trophoblast were transfected with Fugene HD and lipofectamine 2000,positive colonies were detected under fluorescence microscope,and the expression efficiency were measured using flow cytometry analysis between different transfect vectors such as pCAG-EGFP and pEGFP-N3.RESULTS AND CONCLUSION：Expression efficiencies of pCAG-EGFP and pEGFP-N3 were（42.45±3.32）% and（25.95±1.91）% respectively in Fugene HD transfected hESCs,and they had statistically difference（P 0.05）.It was（1.94±0.18）% and（1.49±0.33）% in lipofectamine 2000.The expression efficiencies of pCAG-EGFP and pEGFP-N3 transfected with Fugene HD were higher than that transfected with lipofectamine 2000（P 0.05）.These results indicate that Fugene HD have the greater expression efficiency compared to lipofectamine 2000 in hESC line H9;expression efficiency of EGFP driven by CAG promoter is higher than that by CMV promoter.