Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells

BMC Genomics

Junji Takeda⁶  Kosuke Yusa²  Taroh Kinoshita³  Nakaba Sugimoto⁴  Kyoji Horie¹  Jun Sese⁵  Yusuke Maeda³  Chikara Kokubu⁶  Masahiro Tokunaga⁶ 

[1] Present address: Department of Physiology II, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan;Stem Cell Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK;Laboratory of Immunoglycobiology, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan;Department of Infectious Disease Control, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan;Department of Computer Science, Tokyo Institute of Technology, 2-12-1-W8-60 Oookayama, Meguro-ku, Tokyo 152-8550, Japan;Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

关键词: Whole-exome sequencing;    Saturation mutagenesis;    N-ethyl-N-nitrosourea;    Haploid mouse embryonic stem cell;    GPI-anchor;    Computer simulation;   

Others  :  1091258
DOI  :  10.1186/1471-2164-15-1016

 received in 2014-06-07, accepted in 2014-11-12,  发布年份 2014

【 摘 要 】

Background

Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of “saturation” (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes.

Results

By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation.

Conclusions

Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

【 授权许可】

   
2014 Tokunaga et al.; licensee BioMed Central Ltd.

【 图 表 】

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