@article{2c2573ab11c34e6aa7e26872f9d67273,
title = "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.",
author = "Kyoji Horie and Chikara Kokubu and Junko Yoshida and Keiko Akagi and Ayako Isotani and Akiko Oshitani and Kosuke Yusa and Ryuji Ikeda and Yue Huang and Allan Bradley and Junji Takeda",
note = "Funding Information: We thank K. Kawakami at Japan{\textquoteright}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.",
year = "2011",
month = dec,
doi = "10.1038/nmeth.1739",
language = "English (US)",
volume = "8",
pages = "1071--1081",
journal = "Nature Methods",
issn = "1548-7091",
publisher = "Public Library of Science",
number = "12",
}