Hashemi A, Roohvand F, Ghahremani M H, Aghasadeghi M R, Vahabpour R, Motevali F, Memarnejadian A
1Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
2NRGB, Pasteur institute of Iran, Pasteur Ave., Tehran, Iran.
Cytol Genet. 2012;46(6):347-353. doi: 10.3103/S0095452712060035. Epub 2012 Dec 5.
Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.
高效转染方案的可用性是哺乳动物细胞基因转移研究成功的首要决定因素，针对每种细胞类型都需要通过实验来实现。在此，我们提供了一项比较研究的数据，该研究针对Huh-7和Vero细胞，通过电穿孔和化学方法优化转染条件。针对两种化学试剂jetPEI™和Lipofectamine™ 2000，优化了不同的细胞汇合度、DNA/试剂比例和总转染体积。此外，还研究了电场强度和脉冲长度对提高电穿孔效率的影响。采用pEGFP-N1载体转染细胞，并通过流式细胞术（FACS）和荧光显微镜分析追踪GFP的表达，以此评估转染效率。优化后的电穿孔方案在Huh-7和Vero细胞中的转染率分别为63.73±2.36%和73.9±1.6%，而jetPEI™实现的最大转染水平在Huh-7和Vero细胞中分别为14.2±0.69%和28±1.11%。转染后对细胞进行冷却并没有提高Huh-7细胞的电转染效率。与基于化学的试剂相比，电穿孔在这两种细胞系中均显示出更高的转染水平。所提出的方案应能满足大多数需要对这两种细胞系进行高效转染的实验应用。
