绿色荧光蛋白基因在小鼠胚胎干细胞中表达效率的影响因素分析

目的：研究绿色荧光蛋白（GFP）基因在小鼠胚胎干细胞系R1中表达的影响因素。方法：构建3个不同的GFP真核表达载体。并从转录，蛋白表达水平比较了它们整合于宿主细胞染色体中的表达效率。结果：在GFP蛋白表达水平上，含肽链延长因子（EF）启动子的表达载体和所转染的克隆明显高于含CMV启动子及含双拷贝CMV-GFP表达单元的表达载体；而录水平与蛋白质水平的测定结果相一致。结论：（1）在NIH3T3和R1胚胎干细胞系中，EF启动子引导下的GFP基因表达效率明显高于CMV启动子；（2）外源基因表达单元的拷贝数在染色体中的少量增加，不能明显提高表达效率；（3）获得了在R1胚胎干细胞系中稳定、高效表达GFP的载体，为后续工作奠定了基础。

Analysis of the factors effecting the expression efficiency of the green fluorescent protein gene in mouse embryonic stem cells

Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector pEF GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV GFP with CMV promoter and by pdCMV GFP with double copies of CMV GFP expression unit. There was no significant difference between the colonies transfected by pCMV GFP and pdCMV GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.