A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals

Abstract

Two types of tetracycline-controlled inducible RNAi expression systems have been developed that generally utilize multiple tetracycline operators (TetOs) or repressor fusion proteins to overcome the siRNA leakiness. Here, we report a novel system that overexpresses the tetracycline repressor (TetR) via a bicistronic construct to control siRNA expression. The high level of TetR expression ensures that the inducible promoter is tightly bound, with minimal basal transcription, allowing for regulation solely dependent on TetR rather than a TetR fusion protein via a more complicated mechanism. At the same time, this system contains only a single TetO, thus minimizing the promoter impairment occurring in existing systems due to the incorporation of multiple TetOs, and maximizing the siRNA expression upon induction. In addition, this system combines all the components required for regulation of siRNA expression into a single lentiviral vector, so that stable cell lines can be generated by a single transduction and selection, with significant reduction in time and cost. Taken together, this all-in-one lentiviral vector with the feature of TetR overexpression provides a unique and more efficient tool for conditional gene knockdown that has wide applications. We have demonstrated the high degree of robustness and versatility of this system as applied to several mammalian cells and xenograft animals. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.