1. Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA
2. Department of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
3. Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, Washington 98034, USA
4. These authors contributed equally to this work
5. Present addresses: Department of Biomedical Sciences, Center for Functional Genomics, University at Albany, East Campus, B342A, One University Place, Rensselaer, New York 12144-2345, USA (D.S.C.); Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA (M.S., T.W. and S.J.E.)

Correspondence to: Gregory J. Hannon¹ Email: hannon@cshl.org
Email: selledge@genetics.med.harvard.edu

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^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10}. 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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