A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

Cell
Authors
Keywords
Abstract

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 approximately 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.

Year of Publication
2006
Journal
Cell
Volume
124
Issue
6
Pages
1283-98
Date Published
2006 Mar 24
ISSN
0092-8674
URL
DOI
10.1016/j.cell.2006.01.040
PubMed ID
16564017
Links
Grant list
CA103866 / CA / NCI NIH HHS / United States
P50 CA112962 / CA / NCI NIH HHS / United States
R01CA97061 / CA / NCI NIH HHS / United States