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A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice

Nature Genetics volume 41, pages 946–952 (2009)Cite this article

Abstract

A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.

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Figure 1: Experimental design.
Figure 2: Distribution of LHED insertions.
Figure 3: Surveillance of the intergenic region between Nkx2-2 and Pax1.
Figure 4: Surveillance of the 3′ region of Pax1.

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Acknowledgements

We thank H. Hamada for supporting the initial phase of this study; R. Jaenisch (Massachusetts Institute of Technology) for providing V6.5 ES cells; P.B. Hackett (University of Minnesota) for the pT2/HB and pCMV-SB11 plasmids; and H. Sasaki (RIKEN Center for Developmental Biology) for the pASSHsp68lacZpA plasmid. We also thank M. Kouno, K. Yae, K. Yusa and V.W. Keng for advice and technical assistance. This work was supported by grants from the New Energy and Industrial Technology Development Organization of Japan; RIKEN, the Institute of Physical and Chemical Research; and a grant-in-aid for science research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

  1. Center for Advanced Science and Innovation, Osaka University, Suita, Osaka, Japan

    Chikara Kokubu, Sumi Mizuno & Junji Takeda

  2. Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan

    Chikara Kokubu, Kyoji Horie, Ryuji Ikeda & Junji Takeda

  3. Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan

    Koichiro Abe, Sanae Ogiwara, Masato Ohtsuka & Kenji Imai

  4. Experimental Animal Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan

    Yoshihiro Uno

  5. Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

    Ayako Isotani & Masaru Okabe

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  1. Chikara Kokubu
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Contributions

C.K., K.H., K.I. and J.T. designed the experiments; C.K. and J.T. performed vector construction; C.K., K.H., R.I. and S.M. engineered and analyzed the mouse ES cells; Y.U. generated the knock-in mouse line; A.I. and M.Okabe generated mouse embryos by the tetraploid complementation technique; S.O. and M.Ohtsuka generated mouse embryos by pronuclear injection; C.K. and K.A. analyzed the embryos; K.I. assisted in interpretation of the embryonic data; C.K. performed database analyses; and C.K. wrote the manuscript with assistance from K.H., K.A., M.Okabe and J.T.

Corresponding authors

Correspondence to Chikara Kokubu or Junji Takeda.

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Kokubu, C., Horie, K., Abe, K. et al. A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice. Nat Genet 41, 946–952 (2009). https://doi.org/10.1038/ng.397

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