Single-vector inducible lentiviral RNAi system for oncology target validation

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 addi...