An efficient factor-depleted mammalian in vitro translation system
An efficient factor-depleted mammalian in vitro translation system
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DOI：
10.1038/nprot.2011.314
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发表时间：
2011-05-01
期刊：
影响因子：
14.8
通讯作者：
Hentze, Matthias W.
中科院分区：
文献类型：
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作者：
Rakotondrafara, Aurelie M.;Hentze, Matthias W.
基因表达的大部分调节发生在蛋白质合成水平。除了典型的翻译因子外，许多蛋白质和microRNA（miRNAs）充当调节性反式作用因子。翻译控制的机制分析受益于功能性无细胞系统，该系统可以耗尽负责的调节因子。尽管反义寡核苷酸促进调节RNA的功能性螯合，但蛋白因子的免疫耗竭在技术上具有挑战性。在这里，我们描述了一个简单而强大的替代方案，用于制备来自HeLa细胞的因子耗尽的体外翻译系统。该方法依赖于在提取物制备之前RNA干扰介导的目标因子的敲低，并且它克服了抗体的可用性和特异性以及与所研究的因子相关的蛋白质的共耗尽的问题。完整的程序通常可以在1周内进行，并针对多个（候选）因素并行进行。
Much of the regulation of gene expression occurs at the level of protein synthesis. In addition to the canonical translation factors, a multitude of proteins and microRNAs (miRNAs) act as regulatory trans-acting factors. Mechanistic analysis of translational control benefits from functional cell-free systems that can be depleted of the responsible regulatory factors. Although antisense oligonucleotides facilitate the functional sequestration of the regulatory RNAs, immunodepletion of protein factors is technically challenging. Here we describe a simple and robust alternative protocol for the preparation of factor-depleted in vitro translation system derived from HeLa cells. The procedure relies on RNA interference-mediated knockdown of the factor of interest prior to extract preparation, and it overcomes problems with the availability and specificity of antibodies, as well as with the co-depletion of proteins associated with the factor under study. The complete procedure can normally be conducted within 1 week and carried out in parallel for multiple (candidate) factors.