悬浮细胞电穿孔转染参数的选择研究

目的以凋亡抑制蛋白家族成员Survivin为目的基因,对采用电穿孔转染悬浮培养的小鼠原代淋巴细胞的各种参数进行选择,以提高转染效率。方法通过选择不同的电压、温度、细胞状态等转染参数,采用不同参数组合将化学合成靶向Survivin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,台盼兰拒染试验来评估siRNA的转染率和细胞存活率。结果小鼠原代淋巴细胞在350 V,40μs条件下,可获得最大转染率,约为31%;良好的细胞状态以及低温条件等参数均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 ,通过选择经优化的针对悬浮细胞的转染参数,可以获得较高的转染率。

Optimization of Electroporation Parameters in Suspension Cell

Objective To increase transfection efficiency by choosing the best parameters in mouse primary lymphocyte which cultured in electroporation suspension.Survivin was used as target gene.Methods Specific siRNA targeting Survivin was transfected into mouse primary lymphocyte by using differently electroporation experimental conditions included the voltage,the temperature and the state of cell vitality.The transfection efficiencies were evaluated by light microscope,cell counting and trypan blue staining cell viability assay.Results The highest electroporation transfection rate was achieved under the conditions of voltage 350 V,electric time 40 μs for the mouse lymphocyte.The tansfection efficiencies were closely related to the vigorous state of the cells.The colder operation temperature can bring better transfection efficiency.Conclusion Electroporation might be a highly efficient gene transfection method.Optimized electroporation parameters can improve the transfection efficiency.