An efficient method for generation of bi-allelic null mutant mouse embryonic stem cell lines and its application for investigating epigenetic modifiers. Mus musculus

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60%, and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re targeting the ‘knockout-first’ allele, and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. Overall design: H3K9me3 ChIP-Seq profiling of Setdb1 depleted mouse ES cells

小鼠胚胎干细胞（ES细胞）是研究生物学过程的常用模型系统，但要揭示隐性表型，需同时灭活两个等位基因。本研究依托国际基因敲除小鼠联盟（International Knockout Mouse Consortium, IKMC）的现有资源，开发了一款靶向载体，可用于在具备条件性敲除制备条件的IKMC“敲除首代”ES细胞系中完成第二个等位基因的灭活。我们将该技术应用于多个表观遗传调控因子，以60%的高筛选效率获得了双等位基因靶向克隆；并通过Flp重组酶在两种无效细胞系中恢复基因表达，证实本系统可通过突变表型回复实验验证基因功能的因果关系。我们设计的策略可避免对“敲除首代”等位基因的二次靶向，同时可筛选ES细胞中的必需基因，其中包括组蛋白甲基转移酶Setdb1。为验证实验结果，我们利用本系统的灵活性，实现了他莫昔芬诱导的条件性基因敲除，并同步设置了遗传背景与他莫昔芬效应的对照实验组。敲除Setdb1的ES细胞会出现严重的生长抑制，该抑制效应无法通过外源Nanog表达或在初始多能性“2i”培养基中培养得以挽救，这表明其自我更新缺陷是通过不依赖于多能性调控网络的通路介导的。本研究开发的构建小鼠ES细胞无效突变体的策略可应用于数千个基因，且可复用现有的IKMC中间载体。整体实验设计：Setdb1缺失的小鼠ES细胞的H3K9me3染色质免疫沉淀测序（ChIP-Seq）谱分析。