Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Nat Protoc. 2012 Feb 2;7(2):374-93. doi: 10.1038/nprot.2011.446.
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
RNA 干扰 (RNAi) 是一种非常有效的工具，几乎可以用于研究所有后生动物和真核模式系统中的基因功能。通过表达短发夹 RNA (shRNA)，在小鼠中进行的 RNAi 提供了传统遗传方法不易实现的东西——诱导性和可逆性基因沉默。然而，与 shRNA 转基因株系生产相关的技术可变性迄今为止限制了它们的广泛使用。在这里，我们描述了一种基于 miR30 的 shRNA 转基因小鼠的生成方法，该方法可实现高效且一致地靶向 doxcycline 调控的荧光连接 shRNA 到 Col1a1 基因座。值得注意的是，该方案详细说明了基于 miR30 的 shRNA 的设计和测试中的关键步骤，以最大限度地提高开发有效转基因株系的潜力。总的来说，这个 14 周的程序为任何实验室提供了一种快速且具有成本效益的方法，用于在小鼠体内体内研究基因功能。
