title: Single-vector inducible lentiviral RNAi system for oncology target validation.
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.
authors.affiliation: Oncology Research, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA. dmitri.wiederschain@novartis.com
authors.forename: Dmitri
authors.initials: D
authors.lastname: Wiederschain
authors.forename: Susan
authors.initials: S
authors.lastname: Wee
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Alice
authors.initials: A
authors.lastname: Loo
authors.forename: Guizhi
authors.initials: G
authors.lastname: Yang
authors.forename: Alan
authors.initials: A
authors.lastname: Huang
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Giordano
authors.initials: G
authors.lastname: Caponigro
authors.forename: Yung-Mae
authors.initials: YM
authors.lastname: Yao
authors.forename: Christoph
authors.initials: C
authors.lastname: Lengauer
authors.forename: William R
authors.initials: WR
authors.lastname: Sellers
authors.forename: John D
authors.initials: JD
authors.lastname: Benson
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: C069077:BMI1 protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C489348:PCGF2 protein, human; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1