Abstract

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.

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Acknowledgements

We thank B. Piqani, I. Budianto, D. Szeto, T. Hirozane-Kishikawa, V. Swearingen, A. MacWilliams, T. Nieland, S. Hoang, J. Bochicchio, S. Young, A. Berlin, C. Russ, M. Garber and members of the Broad Institute Genetic Sequencing Platform who provided technical assistance or advice throughout this project, and J. Zhao, T. Roberts and T. Golub for participation in kinase ORFs subcollection generation. This work was supported by Broad Institute Scientific Planning and Allocation of Resources Committee funding, The Ellison Foundation (D.E.H. and M.V.), Dana-Farber Cancer Institute sponsored research funds to CCSB and Center for Cancer Genome Discovery and US National Institutes of Health R33 CA128625 (W.C.H., D.E.R. and D.E.H.).

Author information

Author notes

    • Kourosh Salehi-Ashtiani
    •  & Haley Hieronymus

    Present addresses: New York University Abu Dhabi, Abu Dhabi, United Arab Emirates and Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA (K.S.-A.) and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA (H.H.).

    • Xiaoping Yang
    • , Jesse S Boehm
    • , Xinping Yang
    • , Kourosh Salehi-Ashtiani
    • , Tong Hao
    • , Yun Shen
    •  & Rakela Lubonja

    These authors contributed equally to this work.

Affiliations

  1. RNA interference (RNAi) Platform, Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Xiaoping Yang
    • , Rakela Lubonja
    • , Ozan Alkan
    • , Tashfeen Bhimdi
    • , Thomas M Green
    • , Serena J Silver
    • , Cindy Nguyen
    •  & David E Root
  2. Cancer Program, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Jesse S Boehm
    • , Sapana R Thomas
    • , Cory M Johannessen
    • , Haley Hieronymus
    •  & William C Hahn
  3. Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Xinping Yang
    • , Kourosh Salehi-Ashtiani
    • , Tong Hao
    • , Yun Shen
    • , Ryan R Murray
    • , Dawit Balcha
    • , Changyu Fan
    • , Chenwei Lin
    • , Lila Ghamsari
    • , Marc Vidal
    • , William C Hahn
    •  & David E Hill
  4. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Xinping Yang
    • , Kourosh Salehi-Ashtiani
    • , Tong Hao
    • , Yun Shen
    • , Ryan R Murray
    • , Dawit Balcha
    • , Changyu Fan
    • , Chenwei Lin
    • , Lila Ghamsari
    • , Marc Vidal
    •  & David E Hill
  5. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Xinping Yang
    • , Kourosh Salehi-Ashtiani
    • , Tong Hao
    • , Yun Shen
    • , Ryan R Murray
    • , Dawit Balcha
    • , Changyu Fan
    • , Chenwei Lin
    • , Lila Ghamsari
    • , Marc Vidal
    •  & David E Hill
  6. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Cory M Johannessen
    •  & William C Hahn
  7. Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Cory M Johannessen
    •  & William C Hahn
  8. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Haley Hieronymus

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Contributions

J.S.B., Xia. Y. and D.E.R. wrote the manuscript and, together with D.E.H., K.S.-A. and M.V., designed and supervised the process of creating clonal sequenced ORF collections. Xin. Y., D.B., L.G., J.S.B., S.R.T., H.H., R.R.M., K.S.-A. and C.M.J. generated the starting collections of ORFs. R.L., O.A., J.S.B., C.N., Xia. Y., S.J.S., S.R.T. and C.M.J. created the final libraries, DNA, viruses and pLX vectors, and evaluated expression. T.H., Y.S., C.F., C.L., J.S.B., T.B., T.M.G., Xia. Y. and D.E.R. performed bioinformatic analyses. M.V., W.C.H., D.E.H. and D.E.R. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marc Vidal or William C Hahn or David E Hill or David E Root.

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DOI

https://doi.org/10.1038/nmeth.1638

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