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首页> 外文期刊>Cytology and genetics >Optimization of Transfection Methods for Huh-7 and Vero Cells: A Comparative Study1
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Optimization of Transfection Methods for Huh-7 and Vero Cells: A Comparative Study1

机译：Huh-7和Vero细胞转染方法的优化：比较研究1

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摘要

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~(TM) and Lipofectamine~(Tm) 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N 1 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.
机译：有效转染方案的可用性是成功进行哺乳动物细胞基因转移研究的第一个决定因素，这是针对每种细胞类型通过实验完成的。在此，我们提供了通过电穿孔和化学方法优化Huh-7和Vero细胞转染条件的比较研究数据。针对jetPEI〜（TM）和Lipofectamine〜（Tm）2000这两种化学试剂优化了不同的细胞融合度，DNA /试剂比例和总转染量。此外，还研究了电场强度和脉冲长度的影响以提高电穿孔效率。通过pEGFP-N 1载体转染细胞并通过FACS和荧光显微镜分析追踪GFP的表达是评估转染效率的方法。优化的电穿孔方案分别在Huh-7和Vero细胞中产生63.73±2.36和73.9±1.6％的转染率，而jetPEI可以达到最大转染水平。 Huh-7和Vero细胞分别为14.2±0.69和28±1.11％。细胞的转染后冷却并没有提高Huh-7细胞的电转染效率。与化学试剂相比，电穿孔在两种细胞系中均显示出较高的转染水平。提出的协议应满足大多数需要这两个细胞系转染效率的实验应用。

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  • 来源
    《Cytology and genetics》 |2012年第6期|347-353|共7页
  • 作者

    A. Hashemi; F. Roohvand; M. H. Ghahremani; M. R. Aghasadeghi; R. Vahabpour; F. Motevali; A. Memarnejadian;

  • 作者单位

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran;

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran,NRGB, Pasteur institute of Iran, Pasteur Ave., Tehran;

    Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences;

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran;

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran;

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran,NRGB, Pasteur institute of Iran, Pasteur Ave., Tehran;

    Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran;

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