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Single-vector inducible lentiviral RNAi system for oncology target validation

Wiederschain, Dmitri and Wee, Susan and Chen, Lin and Loo, Alice and Yang, Guizhi and Huang, Alan and Chen, Yan and Caponigro, Giordano and Yao, Yung-Mae and Lengauer, Christoph and Sellers, William and Benson, John (2009) Single-vector inducible lentiviral RNAi system for oncology target validation. Cell Cycle, 8 (3). pp. 498-504. ISSN 1538-4101

Abstract

The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.

Item Type: Article
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Additional Information: Archiving not formally supported by this publisher
Keywords: inducible; single-vector; shRNA; xenograft; cancer; Bmi-1; Mel-18
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Date Deposited: 13 Oct 2015 13:16
Last Modified: 13 Oct 2015 13:16
URI: https://oak.novartis.com/id/eprint/1906

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