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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells
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Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

机译：在单细胞水平上直接可视化siRNA电转移到癌细胞中

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摘要

The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.
机译：RNA干扰介导的基因沉默方法有望基于对疾病相关基因的靶向抑制而用于治疗。电通透化是成功用于在体外和体内将siRNA转移到活细胞中的非病毒方法之一。尽管这种方法在通过RNA干扰进行基因沉默的领域中很有效，但对支持siRNA转移的基本过程知之甚少。在这项研究中，我们通过在单细胞水平上直接可视化研究了Alexa Fluor 546标记的siRNA传递到稳定表达增强型绿色荧光蛋白（EGFP）作为靶基因的鼠类黑色素瘤细胞中。 siRNA的电转移通过延时荧光显微镜进行定量，并与egfp表达的沉默相关。仅在面对阴极的一侧，穿过质膜观察到带负电荷的siRNA直接转移到细胞质中。当在电脉冲之后添加时，siRNA不能有效沉默基因，因为它无法穿透细胞。因此，我们报道电场既作用于细胞质膜的透化作用，又作用于带负电荷的siRNA分子从本体相进入细胞质的电泳阻力。 siRNA的转移动力学与纳米孔的形成兼容，这是用合成纳米孔的技术描述的。所涉及的机制显然对电转移分子的物理化学性质具有特异性，并且与小分子或质粒DNA所观察到的机制不同。

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  • 作者

    A. Paganin-Gioanni; E. Bellard; J. M. Escoffre; M. P. Rols; J. Teissie; M. Golzio;

  • 作者单位

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

    CNRS (Centre National de la Recherche Scientifique), IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France,Universite de Toulouse, UPS (Universite Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    electroporation; transfection; gene therapy; imaging; targeting;

    机译：电穿孔转染基因治疗;成像定位;

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