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Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs

  • Shota Nakade
  • Tetsushi Sakuma
  • Yuto Sakane
  • Yoshihiro Hara
  • Atsushi Kurabayashi
  • Keiko Kashiwagi
  • Akihiko Kashiwagi
  • Takashi Yamamoto
  • Masanobu Obara
Report

Abstract

Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.

Keywords

Homeolog TALEN Targeted mutagenesis Xenopus laevis 

Notes

Acknowledgments

We thank Dr. Ken-Ichi T. Suzuki (Hiroshima University, Japan) for his participation in helpful discussions. The authors wish to express their thanks to Dr. Daniel Voytas (University of Minnesota, St. Paul, MN) for supplying the Golden Gate TALEN and TAL Effector Kit. This study was supported by KAKENHI (25890014) to T.S. from the Japan Society for the Promotion of Science.

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Copyright information

© The Society for In Vitro Biology 2015

Authors and Affiliations

  • Shota Nakade
    • 1
  • Tetsushi Sakuma
    • 1
  • Yuto Sakane
    • 1
  • Yoshihiro Hara
    • 1
  • Atsushi Kurabayashi
    • 2
  • Keiko Kashiwagi
    • 2
  • Akihiko Kashiwagi
    • 2
  • Takashi Yamamoto
    • 1
  • Masanobu Obara
    • 3
  1. 1.Department of Mathematical and Life Sciences, Graduate School of ScienceHiroshima UniversityHigashi-HiroshimaJapan
  2. 2.Institute for Amphibian Biology, Graduate School of ScienceHiroshima UniversityHigashi-HiroshimaJapan
  3. 3.Department of Biological Science, Graduate School of ScienceHiroshima UniversityHigashi-HiroshimaJapan

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