Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi

Abstract

The use of shRNA in vivo to investigate genes involved in proliferation and survival is confounded by the competitive advantage of cells with insufficient shRNA expression. Zuber et al. address this issue with a system that combines Tet-regulated shRNA expression, two fluorescent reporters and robust transactivator production. 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.