正交设计优化电转染CHO细胞的方法

Study on Optimizing the Method of Electrotransfection Applying CHO cell by Orthogonal Design

目的:探讨细胞电穿孔转染的优化方法。方法:按照L9(34)正交表安排各因素水平的细胞电敏感性实验,以台盼蓝染色计数确定细胞存活率,最接近50%存活率为最优方案,根据最优方案将CHO-dhfr-细胞与DNA混匀电穿孔转染以验证正交设计结果。结果:CHO-dhfr-细胞电转染的优化参数为低离子强度Tris-Cl缓冲液、750 V/cm、50μF、电击2次、间隔1 min,各因素对转染的影响大小依次为缓冲液、电场强度、脉冲次数、电容。结论:确定了电穿孔法转染CHO-dhfr-细胞的方法,为进一步应用该细胞株表达重组蛋白打下了基础。

Objective： To explore the optimized method of electrotransfection.Methods： Design the cell electro-susceptibility experiment of the factors and levels according to L 9（3 4） orthogonal layout.Confirm the cell livability by cell counting of trypan blue staining.The best project is the fact that cell livability nearing 50%.Perform the electrotransfection according to the best project in order to validate the result of orthogonal design.Results： The optimized parameter of electrotrans-fection for CHO-dhfr-cells was low ionic strength Tris-Cl buffer,750 V / cm,50 μF,2 times pulse interval 1 min.The weightiness of all factors in electrotransfection in turn was buffer,volt-age,times of pulse,capacitance.Conclusion： Ascertain the method of electrotransfection on CHO-dhfr-cells.