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Genome-scale loss-of-function screening with a lentiviral RNAi library

Nature Methods volume 3, pages 715–719 (2006)Cite this article

Abstract

The discovery that RNA interference (RNAi) is functional in mammalian cells led us to form The RNAi Consortium (TRC) with the goal of enabling large-scale loss-of-function screens through the development of genome-scale RNAi libraries and methodologies for their use. These resources form the basis of a loss-of-function screening platform created at the Broad Institute. Our human and mouse libraries currently contain >135,000 lentiviral clones targeting 27,000 genes. Initial screening efforts have demonstrated that these libraries and methods are practical and powerful tools for high-throughput lentivirus RNAi screens.

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Figure 1: Vector map for the pLKO.1 lentiviral vector.

References

  1. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  2. Brummelkamp, T.R. & Bernards, R. New tools for functional mammalian cancer genetics. Nat. Rev. Cancer 3, 781–789 (2003).

    Article  CAS  Google Scholar 

  3. Hannon, G.J. & Rossi, J.J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378 (2004).

    Article  CAS  Google Scholar 

  4. Moffat, J. & Sabatini, D.M. Building mammalian signalling pathways with RNAi screens. Nat. Rev. Mol. Cell Biol. 7, 177–187 (2006).

    Article  CAS  Google Scholar 

  5. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).

    Article  CAS  Google Scholar 

  6. Brummelkamp, T.R. et al. An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat. Chem. Biol. 2, 202–206 (2006).

    Article  CAS  Google Scholar 

  7. Kolfschoten, I.G. et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849–858 (2005).

    Article  CAS  Google Scholar 

  8. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  Google Scholar 

  9. Ngo, V.N. et al. A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106–110 (2006).

    Article  CAS  Google Scholar 

  10. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    Article  CAS  Google Scholar 

  11. Pelkmans, L. et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78–86 (2005).

    Article  CAS  Google Scholar 

  12. Westbrook, T.F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005).

    Article  CAS  Google Scholar 

  13. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).

    Article  CAS  Google Scholar 

  15. Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

    Article  CAS  Google Scholar 

  17. Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37, 1281–1288 (2005).

    Article  CAS  Google Scholar 

  18. Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    Article  CAS  Google Scholar 

  19. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  20. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  Google Scholar 

  21. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  22. Chen, W., Arroyo, J.D., Timmons, J.C., Possemato, R. & Hahn, W.C. Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity. Cancer Res. 65, 8183–8192 (2005).

    Article  CAS  Google Scholar 

  23. Masutomi, K. et al. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc. Natl. Acad. Sci. USA 102, 8222–8227 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank J. Grenier, G. Hinkle, B. Luo, J. Moffat and S. Silver for their contributions to the work reviewed here and for their comments on the manuscript. The TRC library is the product of The RNAi Consortium (TRC). We thank the members of TRC (Academia Sinica, Bristol-Meyers Squibb, Eli Lilly, Novartis, Sigma-Aldrich, the Broad Institute of MIT and Harvard, Dana-Farber Cancer Institute, Massachusetts General Hospital, Harvard Medical School, the Massachusetts Institute of Technology, and the Whitehead Institute for Biomedical Research) for their scientific contributions and financial support. The Broad Institute and TRC do not profit from the distribution of the TRC library. Sigma-Aldrich, one of five sponsoring members of The RNAi Consortium who contributed equally to funding the work described here, is a commercial distributor of TRC shRNA library clones.

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Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, 02142, Massachusetts, USA

    David E Root, Nir Hacohen, William C Hahn, Eric S Lander & David M Sabatini

  2. Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Nir Hacohen

  3. Harvard Medical School, Boston, 02115, Massachusetts, USA

    Nir Hacohen, William C Hahn & Eric S Lander

  4. Department of Medical Oncology, Center for Cancer Systems Biology, Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    William C Hahn

  5. Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA

    Eric S Lander & David M Sabatini

  6. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Eric S Lander & David M Sabatini

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  1. David E Root
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Correspondence to David E Root.

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Root, D., Hacohen, N., Hahn, W. et al. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods 3, 715–719 (2006). https://doi.org/10.1038/nmeth924

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