Establishment of an Efficient BAC Transgenesis Protocol and its Application to Functional Characterization of the Mouse Brachyury Locus

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  • ABE Kuniya
    Research & Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute Institute of Molecular Embryology and Genetics, Kumamoto University
  • HAZAMA Masaaki
    Department of Biological Sciences, Kyoto University Faculty of Medicine
  • KATOH Hideki
    Experimental animals Institute, Hamamatsu University School of Medicine
  • YAMAMURA Ken-ichi
    Institute of Molecular Embryology and Genetics, Kumamoto University Center for Animal Research and Development, Kumamoto University
  • SUZUKI Misao
    Center for Animal Research and Development, Kumamoto University

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Other Title
  • Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse <italic>Brachyury</italic> locus
  • Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse.

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Description

Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.<br>

Journal

  • Experimental Animals

    Experimental Animals 53 (4), 311-320, 2004

    Japanese Association for Laboratory Animal Science

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