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目的探究采用电穿孔法将质粒pGenesil-1(简称p G)转染老年人成纤维细胞,从中寻找最佳的参数。方法采用电穿孔法将能表达G418抗性蛋白和增强型绿色荧光蛋白(EGFP)的质粒p G转染老年人成纤维细胞。荧光显微镜下观察EGFP表达并测定转染效率、细胞存活率;通过G418筛选得到稳定转染的细胞。结果本实验室电穿孔法介导的老年人成纤维细胞转染的最佳电转条件:电压:240 V;电容:750μF;DNA剂量:8μg。48 h后电穿孔法的转染率接近30%。结论用电穿孔法将质粒pG稳定转染老年人成纤维细胞耗时短,有利于在体外对老年人成纤维细胞进行后续实验。
Objective To explore the use of electroporation plasmid pGenesil-1 (referred to as p G) transfected elderly fibroblasts, looking for the best parameters. METHODS: Plasmid p G, which expressed G418-resistant protein and enhanced green fluorescent protein (EGFP), was transfected into human fibroblasts by electroporation. The expression of EGFP was observed under a fluorescence microscope and the transfection efficiency and the cell survival rate were determined. Stably transfected cells were screened by G418. Results Electroporation in our laboratory was the best condition for electroforming the human fibroblasts. Voltage: 240 V; capacitance: 750 μF; DNA dose: 8 μg. After 48 h electroporation transfection rate close to 30%. Conclusion Electroporation of plasmid pG stably transfected elderly fibroblasts time-consuming, is conducive to in vitro experiments on the elderly fibroblasts.