基于蜕皮激素的诱导性RNA干扰在稳定哺乳动物细胞系中实现基因敲低。

Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines.

作者信息

Rangasamy Danny, Tremethick David J, Greaves Ian K

机构信息

The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, Australian Capital Territory 2601, Australia.

出版信息

Nat Protoc. 2008;3(1):79-88. doi: 10.1038/nprot.2007.456.

DOI:10.1038/nprot.2007.456

PMID:18193024

Abstract

RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.

摘要

RNA干扰（RNAi）是用于哺乳动物基因组中必需基因功能分析的强大工具。在此，我们介绍一种基于蜕皮激素的简单诱导型RNAi方法，该方法可实现高诱导以及对短发夹RNA（shRNA）表达的可调节控制，从而在多种哺乳动物细胞中沉默基因表达。本方案描述了以下内容：诱导型shRNA的设计与克隆；基因敲低的测试与验证；以及建立稳定细胞系的方法。这个逐步的方案为研究细胞周期调控和发育所必需基因的功能提供了一种快速且经济高效的方法，并且可以在不到6周的时间内完成。