电穿孔法高效转染COS-7成纤维细胞系

High efficiency electroporation of COS-7 cell lines

目的 以电穿孔法转染COS 7细胞系以得到较高的转染率。方法 以CMV/LacZ质粒转化COS 7细胞系 ,48小时后用X gal染色。观察不同的穿孔电压 (6 0 0V ,5 5 0V ,5 0 0V ,45 0V ,40 0V)、时间常数 (40us、10 0us)、操作温度 (室温 ,4℃ )、质粒浓度 (5us/ml,2 5us/ml)和细胞悬液体积 (2 0 0ul、40 0ul)对转染率的影响。结果 利用Eppendorf公司的Multiporator及低渗透压缓冲液转化COS 7细胞的最优条件是 :转化体积 40 0 μl,细胞密度 2× 10 6cells/ml,场强 2 0 0 0V/cm ,时间常数 10 0us,室温质粒浓度2 5 μg/ml。条件优化后COS 7转染率可达 85 %。 结论 优化的电穿孔法可得到较高的COS 7转染率。

Objective To optimize the transfection rate in COS 7 cell line with electroporation method. Method CMV/LacZ was transfected into COS 7 cell line by electroporation and X gal staining was Performed 48 hours later. The effect of different poration voltage(600V,550V,500V,450V,400V),time constant(40us,100us),operation temperature(room temperature,4℃),plasmid concentration(5ug/ml,25mg/ml) and transfection volume(200ul,400ul) on transfection rate was observed. Results The transfection rate was affected by voltage,time constant and plasmid concentration,while the survival rate was influenced by voltage,time constant,operation temperature and transfection volume. COS 7 transfection rate was able to reach as high as 85% with optimized variables. Conclusion Optimized electroporation led to a considerably higher COS 7 transfection rate.