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

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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Figure 1: Experimental design.
Figure 2: Regulation of the cNP cassette.
Figure 3: Isolation and characterization of homozygous mutants.
Figure 4: Phenotypic analyses of Dgcr8 and Ptpn11 homozygous mutant cells.
Figure 5: Phenotypic analyses of homozygous mutant ESC lines.

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Acknowledgements

We thank K. Kawakami at Japan's National Institute of Genetics for providing the Tol2 transposon vector; E. Casanova at Ludwig Boltzmann Institute for Cancer Research for the ERT2-iCre fusion vectors; A.F. Stewart at Technische Universitaet Dresden for FLPe and FLPo expression vectors; F. Costantini at Columbia University for the generic Rosa26 targeting vector; T. Sudo and H. Akiyama for microarray analysis; Y. Esaki, Y. Koreeda and M. Okabe for assistance with the production of chimeric mice; M. Araki, K. Araki and K. Yamamura for deposition of ESC clones at the International Gene Trap Consortium; V. Keng for comments on the manuscript; and A. Yamanishi, M. Kawabata, M. Kouno, S. Tanaka, M. Tsunawaki and Y. Kuromi for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Precursory Research for Embryonic Science and Technology (PRESTO) program from the Japan Science and Technology Agency. This work was also supported in part by the Inamori Foundation, Takeda Science Foundation, Kato Memorial Bioscience Foundation and Mochida Memorial Foundation.

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Authors and Affiliations

Authors

Contributions

K.H. designed experiments, constructed vectors, performed ESC culture and phenotypic analyses of mutant ESCs, and wrote the manuscript. C.K. performed bioinformatics analyses and contributed to writing the manuscript. J.Y. conducted vector construction and ESC culture. K.A. performed bioinformatics analyses and constructed the database. A.I. generated chimeric mice. A.O. conducted ESC culture. K.Y. performed gene targeting of ESCs. R.I. conducted ESC culture and PCR genotyping of mutant ESCs. Y.H. and A.B. contributed to the vector design for the selection of homozygous mutants. J.T. conducted vector construction and gene targeting of ESCs, and contributed to writing the manuscript.

Corresponding authors

Correspondence to Kyoji Horie or Junji Takeda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1-5, Supplementary Tables 1-6 (PDF 1599 kb)

Supplementary Data

Vector insertion sites. (XLS 488 kb)

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Horie, K., Kokubu, C., Yoshida, J. et al. A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. Nat Methods 8, 1071–1077 (2011). https://doi.org/10.1038/nmeth.1739

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