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 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.
Wiederschain
Dmitri
D
Oncology Research, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA. dmitri.wiederschain@novartis.com
Wee
Susan
S
Chen
Lin
L
Loo
Alice
A
Yang
Guizhi
G
Huang
Alan
A
Chen
Yan
Y
Caponigro
Giordano
G
Yao
Yung-Mae
YM
Lengauer
Christoph
C
Sellers
William R
WR
Benson
John D
JD
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
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