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

• Published in: Cell 2011-04, Vol.145 (1), p.145-158
• Main Authors: Premsrirut, Prem K., Dow, Lukas E., Kim, Sang Yong, Camiolo, Matthew, Malone, Colin D., Miething, Cornelius, Scuoppo, Claudio, Zuber, Johannes, Dickins, Ross A., Kogan, Scott C., Shroyer, Kenneth R., Sordella, Raffaella, Hannon, Gregory J., Lowe, Scott W.
• Format: Article
• Language: English
• Subjects: adenocarcinoma; Adenocarcinoma - genetics; Adenocarcinoma - therapy; Animals; cost effectiveness; Embryonic Stem Cells - metabolism; Gene Knockdown Techniques - economics; Gene Knockdown Techniques - methods; genes; Lampyridae; Lung Neoplasms - genetics; Lung Neoplasms - therapy; lymphocytic leukemia; lymphoma; Mice; Mice, Transgenic; MicroRNAs - genetics; phenotype; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma - genetics; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma - therapy; Renilla; RNA Interference; RNA Processing, Post-Transcriptional; RNA, Small Interfering - genetics; Signal Transduction; T-lymphocytes; tissues; transgenic animals; Wnt Proteins - metabolism
• ISSN: 0092-8674; 1097-4172
• 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, p16INK4a, p19ARF 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 p19ARF 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. [Display omitted] [Display omitted] ► shRNA transgenics enable potent and reversible fluorescence-marked gene silencing ► Transgenic mouse production is fast, efficient, and scalable ► “Speedy” ES cells accelerate the evaluation of gene function in mouse models ► Reversible gene suppression can pinpoint pathways for therapeutic intervention