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A resource for large-scale RNA-interference-based screens in mammals
Authors:Paddison Patrick J  Silva Jose M  Conklin Douglas S  Schlabach Mike  Li Mamie  Aruleba Shola  Balija Vivekanand  O'Shaughnessy Andy  Gnoj Lidia  Scobie Kim  Chang Kenneth  Westbrook Thomas  Cleary Michele  Sachidanandam Ravi  McCombie W Richard  Elledge Stephen J  Hannon Gregory J
Institution:Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Abstract:Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.
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