Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.
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We thank R. Dickins and C. Miething for discussions in setting up this system, as well as A. Lujambio, A. Rappaport, M. Saborowski and C. Vakoc for testing TRMPV in other models. We also thank Y. Dou (Univ. of Michigan) for providing human MLL-AF9. We gratefully acknowledge B. Ma and S. Muller for excellent technical assistance. We also thank E. Hodges, K. Chang, M. Rooks and the McCombie laboratory for help with Solexa sequencing as well as A. Gordon for bioinformatics support. P. Moody and T. Spencer were of great assistance in flow cytometry. Finally, we thank S. Kogan for histology. This work was supported by the Howard Hughes Medical Institute, the Starr Foundation and the Don Monti Memorial Research Foundation. J.Z. is the Andrew Seligson Memorial Fellow at Cold Spring Harbor Laboratory, and K.M. is the Robert and Teresa Lindsay Fellow of the Watson School of Biological Sciences. L.E.D. is supported by an Overseas Biomedical Research Fellowship of the National Health and Medical Research Council of Australia.
The authors declare no competing financial interests.
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Cancer Cell (2019)
Cell Death & Differentiation (2019)
Cell Reports (2019)