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

Masahiro Tokunaga,Kyoji Horie,Kosuke Yusa,Chikara Kokubu,Nakaba Sugimoto,Taroh Kinoshita,Jun Sese,Yusuke Maeda,Junji Takeda

doi:10.1186/1471-2164-15-1016

Abstract

BackgroundGenome-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.ResultsBy 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1016) contains supplementary material, which is available to authorized users.

Highlights

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 level of “saturation” in mutagenesis has been shown to be a critical parameter for this approach to determine all relevant genes involved in a biological function, without prior knowledge of the gene products [1,2,3]
The cell surface expression of GPI-anchored proteins involves a total of 26 genes, which, with the exception of the X chromosome-linked phosphatidylinositol glycan anchor biosynthesis, class A (Piga) gene, are widely distributed throughout the autosomes (Table 1)

Summary

Introduction

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 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. 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 [3,4,5,6] To overcome these drawbacks, the haploid mouse embryonic stem cell (ESC) system, in which a single-hit mutation can directly lead to phenotypic changes without being compensated by the second copy of the gene, has been recently developed [7,8,9,10], and reviewed in [11].

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Conclusion