A resource for large-scale RNA-interference-based screens in mammals

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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Figure 1: pSHAG-MAGIC shRNA cassette movement strategy.
Figure 2: Microarray analysis of pSHAG-MAGIC library bar codes.
Figure 3: A reverse genetic screen for defects in human proteasome function.
Figure 4: Further validation of selected pSHAG-MAGIC proteasome hairpins.

References

  1. 1

    Hannon, G. J. RNA interference. Nature 418, 244–251 (2002)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Lee, S. S. et al. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nature Genet. 33, 40–48 (2003)

    CAS  Article  Google Scholar 

  4. 4

    Gonczy, P. et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336 (2000)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Fraser, A. G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 (2000)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J. & Conklin, D. S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002)

    CAS  Article  Google Scholar 

  7. 7

    Hemann, M. T. et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nature Genet. 33, 396–400 (2003)

    CAS  Article  Google Scholar 

  8. 8

    Paddison, P. J. & Hannon, G. J. siRNAs and shRNAs: skeleton keys to the human genome. Curr. Opin. Mol. Ther. 5, 217–224 (2003)

    CAS  PubMed  Google Scholar 

  9. 9

    Paul, C. P., Good, P. D., Winer, I. & Engelke, D. R. Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20, 505–508 (2002)

    CAS  Article  Google Scholar 

  10. 10

    Lois, C., Hong, E. J., Pease, S., Brown, E. J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Birrell, G. W., Giaever, G., Chu, A. M., Davis, R. W. & Brown, J. M. A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity. Proc. Natl Acad. Sci. USA 98, 12608–12613 (2001)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Winzeler, E. A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999)

    CAS  Article  Google Scholar 

  14. 14

    Ghoda, L., Sidney, D., Macrae, M. & Coffino, P. Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol. Cell. Biol. 12, 2178–2185 (1992)

    CAS  Article  Google Scholar 

  15. 15

    Chen, P. & Hochstrasser, M. Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86, 961–972 (1996)

    CAS  Article  Google Scholar 

  16. 16

    Heinemeyer, W., Fischer, M., Krimmer, T., Stachon, U. & Wolf, D. H. The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing. J. Biol. Chem. 272, 25200–25209 (1997)

    CAS  Article  Google Scholar 

  17. 17

    Bochtler, M., Ditzel, L., Groll, M., Hartmann, C. & Huber, R. The proteasome. Annu. Rev. Biophys. Biomol. Struct. 28, 295–317 (1999)

    CAS  Article  Google Scholar 

  18. 18

    Coux, O. An interaction map of proteasome subunits. Biochem. Soc. Trans. 31, 465–469 (2003)

    CAS  Article  Google Scholar 

  19. 19

    Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E. & Tansey, W. P. Skp2 regulates Myc protein stability and activity. Mol. Cell 11, 1177–1188 (2003)

    CAS  Article  Google Scholar 

Download references

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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Correspondence to Gregory J. Hannon.

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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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