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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , , & Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463–465 (1989)

  2. 2.

    , & A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 6, 903–918 (1992)

  3. 3.

    et al. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc. Natl Acad. Sci. USA 100, 14109–14114 (2003)

  4. 4.

    International Gene Trap Consortium. A public gene trap resource for mouse functional genomics. Nature Genet. 36, 543–544 (2004)

  5. 5.

    et al. Large-scale gene trapping in C57BL/6N mouse embryonic stem cells. Genome Res. 18, 1670–1679 (2008)

  6. 6.

    , , & Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984)

  7. 7.

    , , & Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445–448 (1986)

  8. 8.

    & Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987)

  9. 9.

    , & Tools for targeted manipulation of the mouse genome. Physiol. Genomics 11, 133–164 (2002)

  10. 10.

    , & Current issues in mouse genome engineering. Nature Genet. 37, 1187–1193 (2005)

  11. 11.

    et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnol. 21, 652–659 (2003)

  12. 12.

    et al. Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly. Proc. Natl Acad. Sci. USA 99, 7548–7553 (2002)

  13. 13.

    et al. Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. Genes Dev. 18, 1553–1564 (2004)

  14. 14.

    . A mouse for all reasons. Cell 128, 9–13 (2007)

  15. 15.

    et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nature Biotechnol. 25, 91–99 (2007)

  16. 16.

    et al. Agouti C57BL/6N embryonic stem cells for mouse genetics resources. Nature Methods 6, 493–495 (2009)

  17. 17.

    et al. Efficient generation of germ line transmitting chimeras from C57BL/6N ES cells by aggregation with outbred host embryos. PLoS ONE 5, e11260 (2010)

  18. 18.

    et al. The IKMC web portal: a central point of entry to data and resources from the International Knockout Mouse Consortium. Nucleic Acids Res. 39 (Database issue). D849–D855 (2011)

  19. 19.

    & Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004)

  20. 20.

    et al. A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles. Genesis 38, 151–158 (2004)

  21. 21.

    et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nature Genet. 28, 241–249 (2001)

  22. 22.

    et al. Caution! Analyze transcripts from conditional knockout alleles. Transgenic Res. 18, 483–489 (2009)

  23. 23.

    , , & Extensive genomic copy number variation in embryonic stem cells. Proc. Natl Acad. Sci. USA 105, 17453–17456 (2008)

  24. 24.

    et al. The vertebrate genome annotation (Vega) database. Nucleic Acids Res. 36, D753–D760 (2008)

  25. 25.

    et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3, 839–844 (2006)

  26. 26.

    et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nature Methods 5, 409–415 (2008)

  27. 27.

    et al. Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res. 10, 116–128 (2000)

  28. 28.

    et al. Feasibility of genome-scale construction of promoter:reporter gene fusions for expression in Caenorhabditis elegans using a multisite gateway recombination system. Genome Res. 14, 2070–2075 (2004)

  29. 29.

    et al. Towards a proteome-scale map of the human protein–protein interaction network. Nature 437, 1173–1178 (2005)

  30. 30.

    , & Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988)

  31. 31.

    et al. Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection. Proc. Natl Acad. Sci. USA 87, 9918–9922 (1990)

  32. 32.

    , , & Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques. Transgenic Res. 7, 135–140 (1998)

  33. 33.

    et al. Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genes. Proc. Natl Acad. Sci. USA 102, 13188–13193 (2005)

  34. 34.

    Two ways to trap a gene in mice. Proc. Natl Acad. Sci. USA 102, 13001–13002 (2005)

  35. 35.

    , , & Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl Acad. Sci. USA 92, 6592–6596 (1995)

  36. 36.

    , , & Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences. Biotechnol. J. 5, 213–223 (2010)

  37. 37.

    , & The length of homology required for gene targeting in embryonic stem cells. Mol. Cell. Biol. 11, 5586–5591 (1991)

  38. 38.

    & Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12, 3365–3371 (1992)

  39. 39.

    , & Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl Acad. Sci. USA 89, 5128–5132 (1992)

  40. 40.

    et al. Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res. 8, 215–221 (1999)

  41. 41.

    et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae. Mol. Cell. Proteomics 6, 439–450 (2007)

  42. 42.

    et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 (2008)

  43. 43.

    et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–1310 (2008)

  44. 44.

    et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998)

  45. 45.

    et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

  46. 46.

    et al. Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray. Genome Biol. 4, R9 (2003)

  47. 47.

    et al. An improved recombineering approach by adding RecA to lambda Red recombination. Mol. Biotechnol. 32, 43–53 (2006)

  48. 48.

    pKSS—a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 138, 109–114 (1994)

Download references


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.).


  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


  1. Search for William C. Skarnes in:

  2. Search for Barry Rosen in:

  3. Search for Anthony P. West in:

  4. Search for Manousos Koutsourakis in:

  5. Search for Wendy Bushell in:

  6. Search for Vivek Iyer in:

  7. Search for Alejandro O. Mujica in:

  8. Search for Mark Thomas in:

  9. Search for Jennifer Harrow in:

  10. Search for Tony Cox in:

  11. Search for David Jackson in:

  12. Search for Jessica Severin in:

  13. Search for Patrick Biggs in:

  14. Search for Jun Fu in:

  15. Search for Michael Nefedov in:

  16. Search for Pieter J. de Jong in:

  17. Search for A. Francis Stewart in:

  18. Search for Allan Bradley in:


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

PDF files

  1. 1.

    Supplementary Information

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

Excel files

  1. 1.

    Supplementary Data

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

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.