电穿孔法基因转染K562细胞条件的优化

Optimization of Electroporation Parameters for K562 Cells DNA transfection

目的以绿色荧光蛋白(GFP)为观察指标优化K562细胞的电穿孔基因转染条件。方法通过改变悬液体积、温度及缓冲液、质粒浓度等转染条件,采用不同条件组合后用电穿孔法将质粒DNA转入K562细胞,通过流式细胞仪和荧光显微镜观察转染效率。结果①在电压250V、电容950μF的条件下.K562细胞得到58.6%的最大转染率。②应用0.4mL细胞悬液体积,当质粒浓度≥20μg/mL时可得到较高的转染效率。③温度和缓冲液中的血清成分对电穿孔效率影响不大。结论电穿孔是一种高效的基因转染法,通过优化条件,可提高转染率。

Objective To optimize the electroporation parameters in K562 cell using GFP （green fluorescent protein） as the observation index. Methods The plasmid DNA was transferred into K562 cell by electroporation using differently experimental conditions including the plasmid density, the volume of cell suspension and the operation temperature. The transfection efficiency was evaluated by flow cytometry（FCM） and fluorescent microscopy. Results The highest transfection rate （58.6% for K562 Cell） was achieved under the condition of voltage 250 V, electric capacity 950 μF. The higher transfection rate was achieved when the volume of cell suspension is 0.4 mL and the plasmid density is more than 20μg/mL. The operation temperature and the concentration of serum in buffer have no effect on the transfection efficiency. Conclusion Electroporation is a highly efficient gene transfection method. The transfection efficiency can be improved by optimizing electroporation parameters.