TY - JOUR
T1 - A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen
AU - Moffat, Jason
AU - Grueneberg, Dorre A.
AU - Yang, Xiaoping
AU - Kim, So Young
AU - Kloepfer, Angela M.
AU - Hinkle, Gregory
AU - Piqani, Bruno
AU - Eisenhaure, Thomas M.
AU - Luo, Biao
AU - Grenier, Jennifer K.
AU - Carpenter, Anne E.
AU - Foo, Shi Yin
AU - Stewart, Sheila A.
AU - Stockwell, Brent R.
AU - Hacohen, Nir
AU - Hahn, William C.
AU - Lander, Eric S.
AU - Sabatini, David M.
AU - Root, David E.
N1 - Funding Information:
This work is a project of the RNAi Consortium (TRC). The TRC was initiated by N.H., W.C.H., E.S.L., D.E.R., D.M.S., S.A.S., and B.R.S. We are grateful to the members of TRC—Academia Sinica, Bristol-Myers Squibb, Eli Lilly, Novartis, and Sigma-Aldrich—for their financial support and scientific advice. We are indebted to Bristol-Myers Squibb, Broad Institute, Dana-Farber Cancer Institute, and Whitehead Institute for Biomedical Research for their support of early phases of this effort. We thank S. Ali, N. Berkowitz, S. Bailey, J. Bridges, L. Brody, S. Bulmer, A. Burds Connor, J. Davies, T.R. Jones, M. Lamprecht, M. Lynes, H. Mizuno, J. Morawiak, C. Nguyen, S. Saif, D. Sarbassov, and S. Yadav for technical assistance; and we are grateful to C. Nusbaum for helpful discussions. We thank J. Evans, A. Davis, the Whitehead-MIT Bioimaging Center, and N. Durso at Cellomics for providing imaging resources and technical advice and Applied Biosystems for providing reagents. This work was also supported by a Dana-Farber/Harvard Cancer Center Core Grant Opportunity Award 2003-31C-NOPA (W.C.H.), NIH P50 CA112962 (W.C.H.), the Tisch Family Fund for Research in Solid Tumors (W.C.H.), an NSERC postdoctoral fellowship (J.M.), NIH CA103866 (D.M.S.), Keck Foundation (D.M.S.), Edith C. Blum Foundation, Stewart Trust (D.M.S.), NIH R01CA97061 (B.R.S), and a Career Award at the Scientific Interface (B.R.S).
PY - 2006/3/24
Y1 - 2006/3/24
N2 - To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and ∼100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.
AB - To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and ∼100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.
UR - http://www.scopus.com/inward/record.url?scp=33646033137&partnerID=8YFLogxK
U2 - 10.1016/j.cell.2006.01.040
DO - 10.1016/j.cell.2006.01.040
M3 - Article
C2 - 16564017
AN - SCOPUS:33646033137
SN - 0092-8674
VL - 124
SP - 1283
EP - 1298
JO - Cell
JF - Cell
IS - 6
ER -