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Floxin, a resource for genetically engineering mouse ESCs

Nature Methods volume 7, pages 50–52 (2010)Cite this article

Abstract

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.

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Figure 1: The Floxin strategy for reversion and modification of gene trap loci.
Figure 2: Efficient reversion of gene trap mutations and Floxin-mediated engineering of new alleles.

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Acknowledgements

We thank K. Thorn for assistance with confocal microscopy, and the members of the Reiter lab for critical reading. Grants from the US National Science Foundation (V.S. and N.S.), US National Institutes of Health (RO1AR054396), California Institute for Regenerative Medicine (RN2-00919), the Burroughs Wellcome Fund, the Packard Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, and the Sandler Family Supporting Foundation (J.F.R.) funded this work. Suz12 and Sall4 cDNAs were gifts from M. Ramalho-Santos (University of California, San Francisco).

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Author notes

  1. Andrew R Norman

    Present address: Present address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA.,

    Veena Singla, Julie Hunkapiller, Nicole Santos, Allen D Seol, Andrew R Norman & Jeremy F Reiter

  2. Department of Human Genetics, University of Chicago, Chicago, Illinois, USA

    Paul Wakenight

  3. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    William C Skarnes

Authors
  1. Veena Singla
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  2. Julie Hunkapiller
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  3. Nicole Santos
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  4. Allen D Seol
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  5. Andrew R Norman
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  6. Paul Wakenight
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  7. William C Skarnes
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  8. Jeremy F Reiter
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Contributions

V.S., J.H., W.C.S. and J.F.R. conceived and designed the experiments. P.W. conceived and designed the first gene trap–Floxin vector. V.S., J.H., N.S., A.D.S., W.C.S., A.R.N. and J.F.R. performed the experiments. J.F.R. and V.S. wrote the paper.

Corresponding authors

Correspondence to William C Skarnes or Jeremy F Reiter.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 2–6 (PDF 3879 kb)

Supplementary Table 1

Floxin-compatible cell lines and corresponding trapped genes. (XLS 683 kb)

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Singla, V., Hunkapiller, J., Santos, N. et al. Floxin, a resource for genetically engineering mouse ESCs. Nat Methods 7, 50–52 (2010). https://doi.org/10.1038/nmeth.1406

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