Screening Methods to Identify TALEN-Mediated Knockout Mice

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Author(s)

    • YAMAMOTO Takashi Yamamoto Takashi
    • Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
    • SUZUKI Ken-Ichi [他] Suzuki Ken-Ichi
    • Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
    • Araki Kimi
    • Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
    • Takeda Naoki
    • Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
    • Ohmuraya Masaki
    • Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
    • Sakuma Tetsushi
    • Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan

Abstract

Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (<i>eGFP</i>) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms.

Journal

  • Experimental Animals

    Experimental Animals 63(1), 79-84, 2014

    Japanese Association for Laboratory Animal Science

Codes

  • NII Article ID (NAID)
    130003391606
  • NII NACSIS-CAT ID (NCID)
    AA11032321
  • Text Lang
    ENG
  • ISSN
    1341-1357
  • NDL Article ID
    025152807
  • NDL Call No.
    Z54-H752
  • Data Source
    NDL  J-STAGE 
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