Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism
Single-cell electroporation of adult sensory neurons for gene screening with RNA interference mechanism
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DOI：
10.1016/j.jneumeth.2008.01.018
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发表时间：
2008-05-30
影响因子：
3
通讯作者：
Scamps, Frederique
中科院分区：
文献类型：
--
作者：
Boudes, Mathieu;Pieraut, Simon;Scamps, Frederique
RNA 干扰似乎是鉴定候选基因或评估基因功能的首选技术。迄今为止，一个主要问题是对天然细胞（例如成体神经元）实现高转染效率。此外，器官或群体培养物的转染不允许接近细胞多样性。背根神经节由多种细胞类型组成，可传递体感感觉。单细胞电穿孔是最新的转染方法，不仅可以将染料或药物引入细胞，还可以将大分子（例如质粒 DNA 表达构建体）引入细胞中。在本研究中，评估了单细胞电穿孔 RNA 干扰技术在成人感觉神经元原代培养中的应用。使用荧光右旋糖酐作为共转染剂，我们首先确定了导致细胞死亡的非特异性 siRNA 浓度。通过肌动蛋白-GFP神经元中GFP荧光的消光以及轴突感觉神经元再生中膜协同转运蛋白Na+-K+-2Cl(-)激活后细胞内Cl-浓度增加的抑制，在蛋白质水平上证明了无毒浓度的siRNA的功效。总之，这些数据表明，通过单细胞电穿孔递送 siRNA 可诱导功能性 RNA 干扰。 (C) 2008 Elsevier B.V. 保留所有权利。
RNA interference appears as a technique of choice to identify gene candidate or to evaluate gene function. To date, a main problem is to achieve high transfection efficiencies on native cells such as adult neurons. In addition, transfection on organ or mass culture does not allow to approach the cellular diversity. Dorsal root ganglia are composed with several cell types to convey somato-sensory sensations. Single-cell electroporation is the most recent method of transfection that allows the introduction into cells, not only dyes or drugs, but also large molecules such plasmid DNA expression constructs. In the present study, the application of the RNA interference technique with the use of single-cell electroporation was evaluated in primary culture of adult sensory neurons. With the use of fluorescent dextran as a co-transfectant, we first determined the non-specific siRNA concentration leading to cell death. Efficacy of siRNA at the non-toxic concentration was demonstrated at the protein level by extinction of GFP fluorescence in actin-GFP neurons and by the inhibition of the intracellular Cl- concentration increase following activation of the membrane co-transporter Na+-K+-2Cl(-) in regenerating axotomized sensory neurons. Altogether, these data show that delivery of siRNAs by single-cell electroporation leads to the induction of functional RNA interference. (C) 2008 Elsevier B.V. All rights reserved.