CHO细胞转染策略的分析及优化方法的建立

Establishment of Optimizing Method and Factor Analysis of Electrotransfection on CHO Cells

按嵌套设计进行细胞电敏感性实验,通过嵌套设计的方差分析了解电穿孔转染缓冲液的pH值、电场强度及电容与活细胞数量的关系。根据嵌套设计结果安排L9(34)正交设计,以最接近50%存活率为期望指标,同时分析主因素间的交互作用并确定最优转染方案。CHO-dhfr-细胞电穿孔转染的优化参数为pH 7.2的低离子强度Tris-Cl缓冲液,525 V/cm、75μF电击2次,间隔1 min。该方法不但确定了电穿孔转染CHO-dhfr-细胞的条件,而且适用于其它种类细胞的电穿孔转染条件的快速优化。

Electrosusceptibility experiment of CHO-dhfr-cells was performed based on to the nested design in order to find out the relation among the pH value of electrotransfection buffer,electric field strength,capacitance and quantity of living cells via ANOVA of nested design.Designed L9（34） orthogonal in accordance with the results of the nested design experiment,specified cell livability nearing 50% is the expectant index.The interaction of primary factors was analyzed and the optimal transfection method was confirmed.The optimal parameters of electrotransfection on CHO-dhfr-cells were electropulse twice on 525 V/cm,75 μF in pH 7.2 low ionic strength Tris-Cl buffer,2 times pulse interval 1 min.The method not only confirms the conditions of electrotransfection on CHO-dhfr-cells but also can apply to the rapidly optimizing the conditions of electrotransfection on other cells.