Abstract

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

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Acknowledgements

We thank the following people for technical assistance: D. Klose, D. Oakley, W. Yang and L. Stebbings for informatics/vector design; R. Bennett, A. Horton and A. van Brunt for manual gene annotation/vector design; L. Cho, R. Li, J.-F. Popoff, M. Sharma and Y. Zhang for recombineering; G. Belteki, P. Tate, Y. Bekele and S. Borchia for targeting vectors; D. Fraser, J. Greystrong, N. Gueorguieva, M. Jackson, P. Ramagiri, I. Walczak, J. Woodward, E. Stebbings, M. Martinez, A. Tsang and Y. Yoshinaga for vector/ES quality control; and D. Edwards, S. Harris, N. Krishnappa, R. Leah and A. Tait for ES cells. We are grateful for advice on the Gateway system from J. Chesnut of Invitrogen. Finally, we wish to thank W. Wurst, K. Lloyd, and our EUCOMM and KOMP colleagues who are contributing to the production and distribution of the conditional knockout resource. This work was funded by the Wellcome Trust Sanger Institute, grants from the National Institutes of Health (KOMP, U01-HG004080 to W.C.S., P.J.d.J. and A.B.) from the EU Sixth Framework Programme (EUCOMM, to W.C.S., A.F.S. and A.B.).

Author information

Author notes

    • Alejandro O. Mujica
    • , Jessica Severin
    •  & Patrick Biggs

    Present addresses: Regeneron Pharmaceuticals, Inc., Tarrytown, New York, USA (A.O.M.); RIKEN Omics Science Center, Yokohama City, Japan (J.S.); Hopkirk Institute, Massey University, New Zealand (P.B.).

Affiliations

  1. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK

    • William C. Skarnes
    • , Barry Rosen
    • , Anthony P. West
    • , Manousos Koutsourakis
    • , Wendy Bushell
    • , Vivek Iyer
    • , Alejandro O. Mujica
    • , Mark Thomas
    • , Jennifer Harrow
    • , Tony Cox
    • , David Jackson
    • , Jessica Severin
    • , Patrick Biggs
    •  & Allan Bradley
  2. Biotechnologisches Zentrum, TU Dresden, 01062 Dresden, Germany

    • Jun Fu
    •  & A. Francis Stewart
  3. Children’s Hospital Oakland Research Institute, Oakland, California 94609, USA

    • Michael Nefedov
    •  & Pieter J. de Jong

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Contributions

W.C.S., B.R., A.P.W, M.K., W.B. and A.O.M. designed the experiments and contributed equally to this work. V.I. and T.C. developed the vector design software. A.O.M., M.T. and J.H. performed and managed manual curation of gene structures and selection of conditional designs. The modular design of targeting vectors was conceived by B.R. Recombineering of vectors was developed by B.R., W.C.S, M.K., M.N. and P.J.d.J., and managed by M.K. and P.J.d.J. Recombineering reagents and advice were supplied by J.F. and A.F.S. High-throughput targeting of ES cells was developed by W.C.S. and managed by W.B. Sequence confirmation of vectors and genotyping of targeted ES cell clones was developed and managed by A.P.W., with informatic support from V.I., D.J., J.S. and P.B. A.B. and A.F.S. inspired the work and wrote the paper together with W.C.S. All authors read and provided comments on the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to William C. Skarnes.

Detailed information on targeted genes is available from the IKMC web portal (http://www.knockoutmouse.org). Targeting constructs and mutant ES cells are available upon request from the EUCOMM (http://www.eummcr.org) and KOMP (http://www.komp.org) repositories.

Supplementary information

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    Supplementary Information

    The file contains Supplementary Tables 1-3, Supplementary Figures 1-6 with legends and additional references.

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    Supplementary Data

    The file contains a list of genes and data for high-throughput gene targeting experiments.

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https://doi.org/10.1038/nature10163

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