Nature Methods
- 3, 707 - 714 (2006)
Published online: 23 August 2006; | doi:10.1038/nmeth923
Lessons from Nature: microRNA-based shRNA librariesKenneth Chang1, Stephen J Elledge2 & Gregory J Hannon11
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. 2
Department of Genetics, Center for Genetics and Genomics, Howard Hughes Medical Institute, Harvard Medical School, Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Correspondence should be addressed to Gregory J Hannon hannon@cshl.org Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.
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