32Dp210细胞稳定转染方法的比较

Stably Transfection of DNA to 32Dp210：A Comparative Study of Three Approaches

目的：采用3种不同的方法将质粒pEGFP-CEBP 转染32Dp210,从中寻找最佳的稳定转染的方法.方法：分别采用阳离子聚合物转染、阳离子脂质体转染和电穿孔转染方法,将能表达G418抗性蛋白和增强型绿色荧光蛋白（EGFP）的质粒pEGFP-CEBP转染32Dp210.24h后,荧光显微镜下观察EGFP表达,流式细胞术测定瞬时转染率;通过G418筛选得到稳定转染的细胞.结果：电转染有最高的瞬时转染效率（58.9%）,稳定转染得到的阳性克隆最多,且从细胞筛选到扩增至得到阳性大克隆所用的时间也最少（21d）;用阳离子聚合物瞬时转染率低（1.7%）,阳离子脂质体转染效果居中,瞬时转染率为5.8%,后面两种方法得到的阳性克隆太少,无法筛选到阳性细胞.结论：用电穿孔转染法将质粒pEGFP-CEBP 转染32Dp210是最佳的转染方法.

Objective To search for the best approaches to transfect plasmid pEGFP--CEBP into 32Dp210. Methods Cationic polymer mediated transfection, cationic liposome transfection and electrotransfection were compared for transfecting plasmid pEGFP-CEBP, a plasmid that expresses G418 resistance protein and Enhanced green fuluorescent protein （EGFP） ,into 32Dp210. The expression of EGFP was observed 24 hours after the trans-feetion by fluorescence microscope. The transient transfection efficiency was measured by flow cytornetry. The stable transfected cells were screened by G418. Results Electrotransfection had the highest transient transfection efficiency（58. 9%）, with the most masc clones of stably transfection and the least time taken to form big masc clones（21 days）. Cationic polymer mediated transient transfeetion and Cationic liposome transfection had the low effi- ciency（1.7% and 5.8 % respectively）, the two methods had few positive clones,it is impossible to get positive cells . Conclusion Electrotransfection is the best way of stably transfeeting plasmid pEGFP--CEBP into 32Dp210.