电穿孔转染悬浮细胞条件的优化

Optimization of Electroporation Parameters for Cell Culture in Suspension

目的以RNA依赖性腺嘌呤脱氨酶(ADAR1)为靶基因优化悬浮培养的小鼠原代淋巴细胞的电穿孔转染条件。方法通过控制电压、温度、细胞状态等转染条件,采用不同条件组合将靶向ADAR1的siRNA导入悬浮培养的小鼠原代淋巴细胞,用倒置显微镜观察、细胞计数、台盼蓝拒染试验来计算siRNA序列转染率和细胞存活率。结果小鼠原代淋巴细胞在400V,40μs条件下可获得最大转染率,约为30.6%;良好的细胞状态以及低温条件可获得更好的转染效果。结论电穿孔转染法是一种高效的基因转染方法,通过优化其转染条件,可以提高其对悬浮细胞的转染率。

Objective To optimize the electroporation parameters in mouse primary lymphocyte cultured in suspension with adenosine deamirase acting on RNA 1 （ADAR1） as a reporter gene. Methods Specific siRNA targeting ADAR1 was transfocted into mouse primary lymphocyte by electropomtion under the different experimental conditions including the voltage, the temperature and the state of cell vitality. The transfection efficiencies were evaluated under the reverse microscope, by cell counting and typan blue staining cell viabihty assay. Results The highest electroporation transfoction rate was achieved under the conditions of voltage 400 V, electric time 40 μs for the mouse lymphocyte. The tansfeetion efficiencies were closely related to the vigorous state of the cells. The colder operation temperature could help better transfection effieieneies. Conclusion Electroporation is a highly efficient gene transfeetion method. Optimized electropomtion parameters can improve the transfection efficiency.