A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen

Jason Moffat, Dorre A. Grueneberg, Xiaoping Yang, So Young Kim, Angela M. Kloepfer, Gregory Hinkle, Bruno Piqani, Thomas M. Eisenhaure, Biao Luo, Jennifer K. Grenier, Anne E. Carpenter, Shi Yin Foo, Sheila A. Stewart, Brent R. Stockwell, Nir Hacohen, William C. Hahn, Eric S. Lander, David M. Sabatini, David E. Root

Research output: Contribution to journalArticlepeer-review

1300 Scopus citations


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.

Original languageEnglish
Pages (from-to)1283-1298
Number of pages16
Issue number6
StatePublished - Mar 24 2006


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