Nature Genetics
36, 183 - 189 (2004)
Published online: 4 January 2004; | doi:10.1038/ng1288
Restriction enzyme−generated siRNA (REGS) vectors and librariesGeorge Sen1, 2, 4, Tom S Wehrman1, 2, 4, Jason W Myers1
& Helen M Blau1, 2, 31
Department of Molecular Pharmacology, 269 Campus Drive, CCSR 4225A Stanford University School of Medicine, Stanford, California 94305, USA. 2
Baxter Laboratory in Genetic Pharmacology, 269 Campus Drive, CCSR 4225A Stanford University School of Medicine, Stanford, California 94305, USA. 3
Department of Microbiology and Immunology, 269 Campus Drive, CCSR 4225A Stanford University School of Medicine, Stanford, California 94305, USA. 4
These authors contributed equally to this work.
Correspondence should be addressed to Helen M Blau hblau@stanford.eduSmall interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme−generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4  105 clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.
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