A Rapid and Scalable System for Studying Gene Function in Mice Using Conditional RNA Interference

Cell. 2011 Apr 1;145(1):145-58. doi: 10.1016/j.cell.2011.03.012.

Abstract

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:

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / genetics
  • Adenocarcinoma / therapy
  • Animals
  • Embryonic Stem Cells / metabolism
  • Gene Knockdown Techniques / economics
  • Gene Knockdown Techniques / methods*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / therapy
  • Mice
  • Mice, Transgenic
  • MicroRNAs / genetics
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / genetics
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / therapy
  • RNA Interference*
  • RNA Processing, Post-Transcriptional
  • RNA, Small Interfering / genetics
  • Signal Transduction
  • Wnt Proteins / metabolism

Substances

  • MicroRNAs
  • RNA, Small Interfering
  • Wnt Proteins