Nature Genetics 37, 1281 - 1288 (2005)
Published online: 2 October 2005; | doi:10.1038/ng1650
Second-generation shRNA libraries covering the mouse and human genomesJose M Silva1, 4, Mamie Z Li2, 4, Ken Chang1, 4, Wei Ge3, Michael C Golding1, Richard J Rickles2, Despina Siolas1, Guang Hu2, Patrick J Paddison1, Michael R Schlabach2, Nihar Sheth1, Jeff Bradshaw3, Julia Burchard3, Amit Kulkarni3, Guy Cavet3, Ravi Sachidanandam1, W Richard McCombie1, Michele A Cleary3, Stephen J Elledge2
& Gregory J Hannon11
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. 2
Department of Genetics, Center for Genetics and Genomics, Brigham and Women's Hospital, Harvard Medical School, Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 3
Rosetta Inpharmatics LLC, 401 Terry Ave. North, Seattle, Washington 98109, USA. 4
These authors contributed equally to this work.
Correspondence should be addressed to Stephen J Elledge selledge@genetics.med.harvard.edu or Gregory J Hannon hannon@cshl.edu Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference–induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.
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