Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902, Japan.
Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science, Wako, Saitama, 351-0198, Japan.
Sci Rep. 2018 Oct 30;8(1):16025. doi: 10.1038/s41598-018-34270-5.
Various molecular biology techniques implementing genome editing have made it possible to generate mouse mutants for nearly all known genes; as a result, the International Mouse Phenotyping Consortium (IMPC) database listing the phenotypes of genetically modified mice has been established. Among mouse phenotypes, lethality is crucial to evaluate the importance of genes in mouse survival. Although many genes are reported to show "preweaning lethality, incomplete penetrance" in the IMPC database, the survival rates of homozygous knockout mice are highly variable. Here, we propose the lethal allele index (LAI), the ratio of the observed number of mice with homozygous knockout (KO) to the theoretically predicted number of homozygous KO mice, as a simple quantitative indicator of preweaning lethality. Among the mice mutants registered as incompletely lethal in IMPC, the LAI calculated from the genotypes of F mice tended to be lower in disease-related genes, and correlated with the frequency of loss-of-function (LOF) alleles in humans. In genome-edited mice using CRISPR/Cas9, the number of mice with homozygous frameshift alleles seemed to be associated with lethality. We edited the Ehd1 gene in cell lines as well as mice using CRISPR/Cas9, and found that the genotype distribution was significantly different. The LAI calculated from these data was similar to the value calculated from the IMPC data. These findings support the potential usefulness of the LAI as an index of preweaning lethality in genome-edited mice.
各种实施基因组编辑的分子生物学技术使得几乎可以为所有已知基因生成小鼠突变体成为可能；因此，建立了国际小鼠表型分析联盟（IMPC）数据库，其中列出了基因修饰小鼠的表型。在小鼠表型中，致死性对于评估基因在小鼠存活中的重要性至关重要。尽管在 IMPC 数据库中报告了许多基因表现出“新生前致死性，不完全外显率”，但纯合敲除小鼠的存活率高度可变。在这里，我们提出致死等位基因指数（LAI），即观察到的纯合敲除（KO）小鼠数量与理论上预测的纯合 KO 小鼠数量的比值，作为评估新生前致死性的简单定量指标。在 IMPC 中注册为不完全致死的小鼠突变体中，从 F 代小鼠的基因型计算得出的 LAI 在与疾病相关的基因中往往较低，并且与人类中功能丧失（LOF）等位基因的频率相关。在使用 CRISPR/Cas9 进行基因组编辑的小鼠中，纯合移码等位基因的小鼠数量似乎与致死性有关。我们使用 CRISPR/Cas9 在细胞系和小鼠中编辑了 Ehd1 基因，并发现基因型分布有显著差异。从这些数据计算出的 LAI 与从 IMPC 数据计算出的值相似。这些发现支持 LAI 作为基因组编辑小鼠新生前致死性指标的潜在有用性。
