人成纤维细胞稳定转染的方法比较

目的采用3种不同的方法将质粒pEGFP-N1转染人成纤维细胞,从中寻找最佳的稳定转染的方法。方法分别采用阳离子聚合物转染、阳离子脂质体转染和电转染方法,将能表达G418抗性蛋白和增强型绿色荧光蛋白(EGFP)的质粒pEGFP-N1转染人成纤维细胞。24h后,荧光显微镜下观察EGFP表达,流式细胞术测定瞬时转染率;通过G418筛选得到稳定转染的细胞。结果电转染有最高的瞬时转染效率(56.8%),稳定转染得到的阳性克隆最多,且从细胞筛选到扩增至得到阳性大克隆所用的时间也最少(约20d);用阳离子聚合物瞬时转染率低(1.7%),稳定筛选同样可以得到阳性大克隆,但是数量很少且从细胞筛选到扩增至阳性大克隆所用的时间也最多(约30d);阳离子脂质体转染效果居中,瞬时转染率为39.9%,从细胞筛选到扩增至得到阳性大克隆所用的时间约25d。结论用电转染法将质粒pEGFP-N1转染人成纤维细胞是最佳的转染方法。

Stably Transfection of DNA to Human Fibroblasts: A Comparative Study of Three Approaches

Objective To search for the best approaches to transfect plasmid pEGFP-N1 into human fibroblasts.Methods Cationic polymer mediated transfection,cationic liposome transfection and electrotransfection were compared for transfecting plasmid pEGFP-N1,a plasmid that expresses G418 resistance protein and Enhanced green fuluorescent protein(EGFP),into Human fibroblasts.The expression of EGFP was observed 24 hours after the transfection by fluorescence microscope.The transient transfection efficiency was measured by flow cytometry.The stable transfected cells were screened by G418.Results Electrotransfection had the highest transient transfection efficiency(56.8%),with the most masc clones of stably transfection and the least time taken to form big masc clones(20 days).Cationic polymer mediated transient transfection had the lowest efficiency(1.7%),with the least masc clones and the longest time taken to form the big masc clones(30 days).Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones.Conclusion Electrotransfection is the best way of stably transfecting plasmid pEGFP-N1 into human fibroblasts.