A resource for large-scale RNA-interference-based screens in mammals (original) (raw)

Nature volume 428, pages 427–431 (2004)Cite this article

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 tool1,2,3,4,5,6,7,8,9,10. 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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Acknowledgements

We thank T. Moore and B. Simmons from Open Biosystems for their help in organizing and rearraying the library, and colleagues at CSHL and elsewhere (as indicated in Supplementary Table 1) as well as J. LaBaer and C. Perou for curating gene lists. G. Katari and J. Faith helped with bioinformatic analysis and shRNA choice, and members of the Lowe laboratory (CSHL) provided advice on vector optimization. This work was supported by an Innovator Award from the US Army Breast Cancer Research Program (G.J.H.), a contract from the National Cancer Institute (G.J.H.), grants from the NIH (G.J.H., W.R.M., S.J.E.) and the US Army Breast Cancer Research Program (G.J.H., D.S.C.), the Howard Hughes Medical Institute (S.J.E.), and by generous support from Oncogene Sciences and Merck. P.J.P. is an Arnold and Mabel Beckman Fellow of the Watson School of Biological Sciences and is supported by a predoctoral fellowship from the US Army Breast Cancer Research Program. J.M.S. is supported by a postdoctoral fellowship from the US Army Prostate Cancer Research Program. S.J.E. is an Investigator of the Howard Hughes Medical Institute. G.J.H. is a Rita Allen Foundation Fellow.

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Author notes

  1. Douglas S. Conklin
    Present address: Department of Biomedical Sciences, Center for Functional Genomics, University at Albany, East Campus, B342A, One University Place, Rensselaer, New York, 12144-2345, USA
  2. Mike Schlabach, Thomas Westbrook & Stephen J. Elledge
    Present address: Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School, Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts, 02115, USA
  3. Patrick J. Paddison, Jose M. Silva and Douglas S. Conklin: These authors contributed equally to this work

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA
    Patrick J. Paddison, Jose M. Silva, Douglas S. Conklin, Shola Aruleba, Vivekanand Balija, Andy O'Shaughnessy, Lidia Gnoj, Kim Scobie, Kenneth Chang, Ravi Sachidanandam, W. Richard McCombie & Gregory J. Hannon
  2. Department of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA
    Mike Schlabach, Mamie Li, Thomas Westbrook & Stephen J. Elledge
  3. Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, Washington, 98034, USA
    Michele Cleary

Authors

  1. Patrick J. Paddison
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  2. Jose M. Silva
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  3. Douglas S. Conklin
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  4. Mike Schlabach
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  5. Mamie Li
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  6. Shola Aruleba
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  7. Vivekanand Balija
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  8. Andy O'Shaughnessy
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  9. Lidia Gnoj
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  10. Kim Scobie
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  11. Kenneth Chang
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  12. Thomas Westbrook
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  13. Michele Cleary
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  14. Ravi Sachidanandam
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  15. W. Richard McCombie
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  16. Stephen J. Elledge
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  17. Gregory J. Hannon
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Corresponding author

Correspondence toGregory J. Hannon.

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The authors declare that they have no competing financial interests.

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Paddison, P., Silva, J., Conklin, D. et al. A resource for large-scale RNA-interference-based screens in mammals.Nature 428, 427–431 (2004). https://doi.org/10.1038/nature02370

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