利用条件性 RNA 干扰快速、可扩展地研究小鼠中的基因功能。
A rapid and scalable system for studying gene function in mice using conditional RNA interference.
机构信息
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
出版信息
Cell. 2011 Apr 1;145(1):145-58. doi: 10.1016/j.cell.2011.03.012.
RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:
RNA 干扰是研究基因功能的有力工具，然而，可重复产生 RNAi 转基因小鼠仍然是一个重大限制。通过将优化的荧光偶联 miR30 基 shRNA 与高效 ES 细胞靶向相结合，我们开发了一种用于生产 shRNA 转基因小鼠的快速、可扩展的流水线。使用该系统，我们生成了八个 tet 调控的 shRNA 转基因系，靶向萤火虫和海肾荧光素酶、Oct4 和肿瘤抑制因子 p53、p16(INK4a)、p19(ARF)和 APC，并在体内广泛的组织中证明了强大的基因沉默和 GFP 跟踪敲低。此外，使用靶向 APC 的 shRNA，我们说明了如何通过这种方法来鉴定已研究基因的预测表型和未知功能。此外，通过调控基因沉默，我们验证 APC/Wnt 和 p19(ARF)分别是 T 细胞急性淋巴细胞白血病/淋巴瘤和肺腺癌的潜在治疗靶点。该系统为生产针对任何哺乳动物基因的 RNAi 转基因小鼠提供了一种具有成本效益和可扩展性的平台。
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