一种用磷酸钙法高效转染HEK293T细胞方法的建立

Establishment of a High-Efficiency Calcium Phosphate Transfection Method for HEK 293T Cells

利用磷酸钙转染法将lacZ-pShuttle质粒导入HEK 293T细胞中,实验证明,pH值、DNA纯度、转染后培养时间和甘油休克时间是影响转染效率的重要因素。转染后培养6h进行甘油休克效果最佳,比培养0h直接进行休克转染效率升高584.4%;甘油休克时间为4.5min时转染效果最佳,比不用甘油休克转染效率升高130.8%。通过对影响转染的几个条件进行优化,建立了1种可以高效转染HEK 293T细胞的方法,且简便易行。

The HEK 293T cells were transfected by calcium phosphate method by using lacZ-pShuttle plasmid DNA, which inserted a lacZ reporter gene in the MCS of the pShuttle plasmid. The expression of lacZ gene was measured with 13-galactosidase protein dyed by X-gal, which revealed the efficiency of transfection. The influences of the pH value, the purity of the plasmid DNA, the cultured time before shocking and the glycerol-shock time to the transfection efficiency were evaluated. The results show that, the best cultured time before shocking is 6 hours, which makes the transfection efficiency increase by 584.4% and the best time of glycerol-shock is 4.5 minutes, making the efficiency increase by 130.8 % compared to the negative group. The pH value of 6.96 and the pure plasmid DNA are also a key factor, affecting the efficiency of transfection significantly. A high-efficiency calcium phosphate transfection method is established by optimizing several transfection conditions. Its efficiency is much higher than the methods which have been reported previously, and it is very easy to use and stable.