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

Kyoji Horie, Chikara Kokubu, Junko Yoshida, Keiko Akagi, Ayako Isotani, Akiko Oshitani, Kosuke Yusa, Ryuji Ikeda, Yue Huang, Allan Bradley, Junji Takeda

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1071-1081
Number of pages11
JournalNature Methods
Volume8
Issue number12
DOIs
StatePublished - Dec 1 2011

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Genetic Testing
Embryonic Stem Cells
Stem cells
Mutagenesis
Screening
Genes
Bloom Syndrome
Genome
Phenotype
Puromycin
Homozygote
Tetracycline
Diploidy
Single Nucleotide Polymorphism
Polymorphism
Population
Nucleotides
Cells
Mouse Embryonic Stem Cells
antibiotic G 418

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Horie, K., Kokubu, C., Yoshida, J., Akagi, K., Isotani, A., Oshitani, A., ... Takeda, J. (2011). A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. Nature Methods, 8(12), 1071-1081. https://doi.org/10.1038/nmeth.1739

A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. / Horie, Kyoji; Kokubu, Chikara; Yoshida, Junko; Akagi, Keiko; Isotani, Ayako; Oshitani, Akiko; Yusa, Kosuke; Ikeda, Ryuji; Huang, Yue; Bradley, Allan; Takeda, Junji.

In: Nature Methods, Vol. 8, No. 12, 01.12.2011, p. 1071-1081.

Research output: Contribution to journalArticle

Horie, K, Kokubu, C, Yoshida, J, Akagi, K, Isotani, A, Oshitani, A, Yusa, K, Ikeda, R, Huang, Y, Bradley, A & Takeda, J 2011, 'A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening', Nature Methods, vol. 8, no. 12, pp. 1071-1081. https://doi.org/10.1038/nmeth.1739
Horie, Kyoji ; Kokubu, Chikara ; Yoshida, Junko ; Akagi, Keiko ; Isotani, Ayako ; Oshitani, Akiko ; Yusa, Kosuke ; Ikeda, Ryuji ; Huang, Yue ; Bradley, Allan ; Takeda, Junji. / A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. In: Nature Methods. 2011 ; Vol. 8, No. 12. pp. 1071-1081.
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