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