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A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening

  1. Author:
    Horie, K.
    Kokubu, C.
    Yoshida, J.
    Akagi, K.
    Isotani, A.
    Oshitani, A.
    Yusa, K.
    Ikeda, R.
    Huang, Y.
    Bradley, A.
    Takeda, J.

  2. Author Address

    [Horie, Kyoji; Kokubu, Chikara; Yoshida, Junko; Yusa, Kosuke; Ikeda, Ryuji; Takeda, Junji] Osaka Univ, Grad Sch Med, Dept Social & Environm Med, Suita, Osaka, Japan. [Horie, Kyoji] Japan Sci & Technol Agcy, Kawaguchi, Saitama, Japan. [Kokubu, Chikara; Oshitani, Akiko; Takeda, Junji] Osaka Univ, Ctr Adv Sci & Innovat, Suita, Osaka, Japan. [Akagi, Keiko] NCI, Mouse Canc Genet Program, Ctr Canc Res, Frederick, MD 21701 USA. [Akagi, Keiko] Ohio State Univ, Ctr Comprehens Canc, Columbus, OH 43210 USA. [Isotani, Ayako] Osaka Univ, Res Inst Microbial Dis, Suita, Osaka, Japan. [Yusa, Kosuke; Huang, Yue; Bradley, Allan] Wellcome Trust Sanger Inst, Cambridge, England. [Huang, Yue] Peking Union Med Coll, Dept Med Genet, Inst Basic Med Sci, Natl Lab Med Mol Biol, Beijing 100021, Peoples R China. [Huang, Yue] Chinese Acad Med Sci, Beijing 100730, Peoples R China.;Horie, K (reprint author), Osaka Univ, Grad Sch Med, Dept Social & Environm Med, Suita, Osaka, Japan;horie@mr-envi.med.osaka-u.ac.jp takeda@mr-envi.med.osaka-u.ac.jp
    1. Year: 2011
    2. Date: Dec
  2. Journal: Nature Methods
    1. 8
    2. 12
    3. Pages: 1071-+
  4. Type of Article: Article
  5. ISSN: 1548-7091
  1. Abstract:

    Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.

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External Sources

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  2. DOI: 10.1038/nmeth.1739
  3. View record in Web of Science®
  4. WOS: 000297931700021

Library Notes

  1. Fiscal Year: FY2011-2012
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