Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Toward simpler and faster genome-wide mutagenesis in mice

Nature Genetics volume 39, pages 922–930 (2007)Cite this article

Abstract

Here we describe a practical Cre-loxP and piggyBac transposon–based mutagenesis strategy to systematically mutate coding sequences and/or the vast noncoding regions of the mouse genome for large-scale functional genomic analysis. To illustrate this approach, we first created loxP-containing loss-of-function alleles in the protocadherin α, β and γ gene clusters (Pcdha, Pcdhb and Pcdhg). Using these alleles, we show that, under proper guidance, Cre-loxP site-specific recombination can mediate efficient trans-allelic recombination in vivo, facilitating the generation of large germline deletions and duplications including deletions of Pcdha, and Pcdha to Pcdhb, simply by breeding (that is, at frequencies of 5.5%–21.6%). The same breeding method can also generate designed germline translocations between nonhomologous chromosomes at unexpected frequencies of greater than 1%. By incorporating a piggyBac transposon to insert and to distribute loxP sites randomly throughout the mouse genome, we present a simple but comprehensive method for generating genome-wide deletions and duplications, in addition to insertional loss-of-function and conditional rescue alleles, again simply by breeding.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generation of mouse Pcdh alleles.
Figure 2: Generation of large deletions and duplications by Cre-loxP–mediated trans-allelic recombination.
Figure 3: Generation of a Cre-loxP–mediated germline translocation between nonhomologous chromosomes.
Figure 4: Optimization of piggyBac for multipurpose gene-trapping.
Figure 5: Germline transposition.
Figure 6: Gene-trap alleles recapitulate a conventional knockout phenotype.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Austin, C.P. et al. The knockout mouse project. Nat. Genet. 36, 921–924 (2004).

    Article  CAS  Google Scholar 

  2. Auwerx, J. et al. The European dimension for the mouse genome mutagenesis program. Nat. Genet. 36, 925–927 (2004).

    Article  CAS  Google Scholar 

  3. The International Mouse Knockout Consortium. A mouse for all reasons. Cell 128, 9–13 (2007).

  4. Dermitzakis, E.T., Reymond, A. & Antonarakis, S.E. Conserved non-genic sequences — an unexpected feature of mammalian genomes. Nat. Rev. Genet. 6, 151–157 (2005).

    Article  CAS  Google Scholar 

  5. Bejerano, G. et al. Ultraconserved elements in the human genome. Science 304, 1321–1325 (2004).

    Article  CAS  Google Scholar 

  6. Boffelli, D., Nobrega, M.A. & Rubin, E.M. Comparative genomics at the vertebrate extremes. Nat. Rev. Genet. 5, 456–465 (2004).

    Article  CAS  Google Scholar 

  7. Sandelin, A. et al. Arrays of ultraconserved non-coding regions span the loci of key developmental genes in vertebrate genomes. BMC Genomics 5, 99 (2004).

    Article  Google Scholar 

  8. Margulies, E.H. et al. Comparative sequencing provides insights about the structure and conservation of marsupial and monotreme genomes. Proc. Natl. Acad. Sci. USA 102, 3354–3359 (2005).

    Article  CAS  Google Scholar 

  9. Vavouri, T., McEwen, G.K., Woolfe, A., Gilks, W.R. & Elgar, G. Defining a genomic radius for long-range enhancer action: duplicated conserved non-coding elements hold the key. Trends Genet. 22, 5–10 (2006).

    Article  CAS  Google Scholar 

  10. Bejerano, G., Haussler, D. & Blanchette, M. Into the heart of darkness: large-scale clustering of human non-coding DNA. Bioinformatics 20, I40–I48 (2004).

    Article  CAS  Google Scholar 

  11. Golic, K.G. & Golic, M.M. Engineering the Drosophila genome: chromosome rearrangements by design. Genetics 144, 1693–1711 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ryder, E. et al. The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics 167, 797–813 (2004).

    Article  CAS  Google Scholar 

  13. Zheng, B., Sage, M., Sheppeard, E.A., Jurecic, V. & Bradley, A. Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol. Cell. Biol. 20, 648–655 (2000).

    Article  CAS  Google Scholar 

  14. Mills, A.A. & Bradley, A. From mouse to man: generating megabase chromosome rearrangements. Trends Genet. 17, 331–339 (2001).

    Article  CAS  Google Scholar 

  15. Herault, Y., Rassoulzadegan, M., Cuzin, F. & Duboule, D. Engineering chromosomes in mice through targeted meiotic recombination (TAMERE). Nat. Genet. 20, 381–384 (1998).

    Article  CAS  Google Scholar 

  16. Kmita, M., Fraudeau, N., Herault, Y. & Duboule, D. Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs. Nature 420, 145–150 (2002).

    Article  CAS  Google Scholar 

  17. Zakany, J. & Duboule, D. Hox genes and the making of sphincters. Nature 401, 761–762 (1999).

    Article  CAS  Google Scholar 

  18. Genoud, N. et al. Disruption of Doppel prevents neurodegeneration in mice with extensive Prnp deletions. Proc. Natl. Acad. Sci. USA 101, 4198–4203 (2004).

    Article  CAS  Google Scholar 

  19. Spitz, F., Herkenne, C., Morris, M.A. & Duboule, D. Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes. Nat. Genet. 37, 889–893 (2005).

    Article  CAS  Google Scholar 

  20. Ding, S. et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473–483 (2005).

    Article  CAS  Google Scholar 

  21. Wu, Q. & Maniatis, T. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97, 779–790 (1999).

    Article  CAS  Google Scholar 

  22. Wu, Q. et al. Comparative DNA sequence analysis of mouse and human protocadherin gene clusters. Genome Res. 11, 389–404 (2001).

    Article  CAS  Google Scholar 

  23. Wu, Q. Comparative genomics and diversifying selection of the clustered vertebrate protocadherin genes. Genetics 169, 2179–2188 (2005).

    Article  CAS  Google Scholar 

  24. Kohmura, N. et al. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20, 1137–1151 (1998).

    Article  CAS  Google Scholar 

  25. Wang, X. et al. Gamma protocadherins are required for survival of spinal interneurons. Neuron 36, 843–854 (2002).

    Article  CAS  Google Scholar 

  26. Phillips, G.R. et al. Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons. J. Neurosci. 23, 5096–5104 (2003).

    Article  CAS  Google Scholar 

  27. Weiner, J.A., Wang, X., Tapia, J.C. & Sanes, J.R. Gamma protocadherins are required for synaptic development in the spinal cord. Proc. Natl. Acad. Sci. USA 102, 8–14 (2005).

    Article  CAS  Google Scholar 

  28. Hambsch, B., Grinevich, V., Seeburg, P.H. & Schwarz, M.K. γ-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression. J. Biol. Chem. 280, 15888–15897 (2005).

    Article  CAS  Google Scholar 

  29. Tang, S.H., Silva, F.J., Tsark, W.M. & Mann, J.R. A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ. Genesis 32, 199–202 (2002).

    Article  CAS  Google Scholar 

  30. Bonin, C.P. & Mann, R.S. A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila. Genetics 167, 1801–1811 (2004).

    Article  CAS  Google Scholar 

  31. Thibault, S.T. et al. A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat. Genet. 36, 283–287 (2004).

    Article  CAS  Google Scholar 

  32. Schmidt, E.E., Taylor, D.S., Prigge, J.R., Barnett, S. & Capecchi, M.R. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl. Acad. Sci. USA 97, 13702–13707 (2000).

    Article  CAS  Google Scholar 

  33. DeChiara, T.M. et al. Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development. Nat. Genet. 24, 271–274 (2000).

    Article  CAS  Google Scholar 

  34. Komori, T. et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764 (1997).

    Article  CAS  Google Scholar 

  35. Jung, J.Y. et al. Expression of urea transporters in potassium-depleted mouse kidney. Am. J. Physiol. Renal Physiol. 285, F1210–F1224 (2003).

    Article  CAS  Google Scholar 

  36. Forster, A. et al. Engineering de novo reciprocal chromosomal translocations associated with Mll to replicate primary events of human cancer. Cancer Cell 3, 449–458 (2003).

    Article  CAS  Google Scholar 

  37. Zong, H., Espinosa, J.S., Su, H.H., Muzumdar, M.D. & Luo, L. Mosaic analysis with double markers in mice. Cell 121, 479–492 (2005).

    Article  CAS  Google Scholar 

  38. Dracopoli, N.C. et al. in Current Protocols in Human Genetics 4.1.1–4.1.4 (John Wiley & Sons, Hoboken, New Jersey, 2006).

    Google Scholar 

  39. Cary, L.C. et al. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156–169 (1989).

    Article  CAS  Google Scholar 

  40. Fraser, M.J., Ciszczon, T., Elick, T. & Bauser, C. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol. Biol. 5, 141–151 (1996).

    Article  CAS  Google Scholar 

  41. Li, X. et al. piggyBac internal sequences are necessary for efficient transformation of target genomes. Insect Mol. Biol. 14, 17–30 (2005).

    Article  Google Scholar 

  42. Friedrich, G. & Soriano, P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523 (1991).

    Article  CAS  Google Scholar 

  43. Liu, P., Jenkins, N.A. & Copeland, N.G. Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells. Nat. Genet. 30, 66–72 (2002).

    Article  CAS  Google Scholar 

  44. Handler, A.M. & Harrell, R.A. II Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP. Insect Biochem. Mol. Biol. 31, 199–205 (2001).

    Article  CAS  Google Scholar 

  45. Boulet, A.M. & Capecchi, M.R. Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod. Development 131, 299–309 (2004).

    Article  CAS  Google Scholar 

  46. Economides, K.D., Zeltser, L. & Capecchi, M.R. Hoxb13 mutations cause overgrowth of caudal spinal cord and tail vertebrae. Dev. Biol. 256, 317–330 (2003).

    Article  CAS  Google Scholar 

  47. Bunting, M., Bernstein, K.E., Greer, J.M., Capecchi, M.R. & Thomas, K.R. Targeting genes for self-excision in the germ line. Genes Dev. 13, 1524–1528 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.R. Mann (City of Hope's Beckman Research Institute) for the Hprt-Cre mouse; J.-F. Cheng (Lawrence Berkeley National Laboratory) for the Pcdh BAC clones; C. Zou and H. Peng (laboratory of Q.W.) for constructing Pcdha1 and Pcdhga1 targeting vectors and performing DNA blot experiments; M. Hockin for help with chromosome painting; G. Karan for fluorescence photography of adult mice; members of M.R.C.'s laboratory for comments on the manuscript and the University of Utah Transgenic/Gene Targeting Facility for pronuclear injections. pBigT was a gift of F. Constantini (Columbia University), plasmid C4-PBss was gift of R.S. Mann (Columbia University) and plasmid 286 was a gift of A. Handler (University of Florida). We are grateful for technical support from the ES cell culture, mouse surgery and husbandry staff in M.R.C.'s laboratory, in particular S. Barnett, L. Byers, C. Lenz, K. Lustig, J. Tomlin and J. Shuhua. G.Y. and Q.W. were supported by a grant from the American Cancer Society. Q.W. is a Basil O'Connor Scholar.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of Utah, Salt Lake City, 84112, Utah, USA

    Sen Wu & Mario R Capecchi

  2. Department of Human Genetics, University of Utah, Salt Lake City, 84112, Utah, USA

    Guoxin Ying, Qiang Wu & Mario R Capecchi

Authors
  1. Sen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guoxin Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mario R Capecchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mario R Capecchi.

Ethics declarations

Competing interests

A patent application has been filed by the University of Utah that includes some of the work presented in this publication.

Supplementary information

Supplementary Fig. 1

Gene targeting strategy for the Pcdha1eGFP, Pcdhac1AP, Pcdhac2lacZ, PcdhaCon and Pcdhga1hrGFP knockout alleles. (PDF 224 kb)

Supplementary Fig. 2

Normal gross anatomy in the brain of an adult del(α)/del(α) mouse. (PDF 797 kb)

Supplementary Fig. 3

Variations of ZG-s gene-trap vectors. (PDF 313 kb)

Supplementary Fig. 4

X-gal staining of more examples of gene-trap alleles and somatic transallelic recombination using gene-trap loxP alleles. (PDF 277 kb)

Supplementary Table 1

Genotyping primers for all alleles in this study. (PDF 101 kb)

Supplementary Table 2

piggyBac transpositions in founder mice. (PDF 17 kb)

Supplementary Table 3

piggyBac transpositions in the germline through breeding. (PDF 20 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, S., Ying, G., Wu, Q. et al. Toward simpler and faster genome-wide mutagenesis in mice. Nat Genet 39, 922–930 (2007). https://doi.org/10.1038/ng2060

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng2060

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing