An inducible and reversible embryonic stem cell biobank reveals functional genomic pathways and disease targets [RNA-Seq]

Clonal cellular variance often confounds reproducibility of forward and reverse genetic studies. We developed combinatorial approaches for whole genome saturated mutagenesis using haploid murine ES cells to permit induction and reversion of genetic mutations. Using these systems, we created a biobank with over 100000 individual ES cell lines with repairable and genetically bar coded mutations targeting 16950 genes. This biobank termed “Haplobank” is freely available. In addition, we developed a genetic color coding system for rapid repair of mutations and direct functional validation in sister clones. Using this system, we report functional validation of essential ES cell genes. We also identified phospholipase16G as a key pathway for cytotoxicity of human rhinoviruses, the most frequent cause of the common cold. Moreover, we derived 3D blood vessel organoids from haploid ES cells, combining conditional mutagenesis in haploid ES cells with tissue engineering. We identified multiple novel genes, such as Connexin43/Gja1, in blood vessel formation and tip cell specification in vitro and also in vivo. Taken together, we develop a conditional homozygous ES cell resource for the community to empower controlled genetic studies in murine ES cells and tissues derived from it.

克隆细胞异质性常常干扰正向与反向遗传学研究的可重复性。我们借助单倍体小鼠胚胎干细胞（ES cells）开发了全基因组饱和诱变的组合策略，以实现遗传突变的诱导与回复。依托该系统，我们构建了包含超10万株独立胚胎干细胞系的生物样本库，每株细胞均携带可修复且经基因条形码标记的突变，覆盖16950个基因。这款被命名为"单倍体库（Haplobank）"的生物样本库可免费获取。此外，我们开发了一套基因色彩编码系统，可实现突变的快速修复以及姐妹克隆的直接功能验证。借助该系统，我们完成了关键胚胎干细胞基因的功能验证。我们还发现磷脂酶16G（phospholipase 16G）是引发普通感冒的最常见病原体——人鼻病毒的细胞毒性关键通路。进一步，我们将单倍体胚胎干细胞的条件诱变技术与组织工程相结合，从单倍体胚胎干细胞中构建出三维血管类器官。我们在体外与体内环境中均鉴定出多个参与血管生成与顶端细胞特化的新基因，例如连接蛋白43/Gja1（Connexin43/Gja1）。综上，我们开发了一套条件纯合胚胎干细胞资源库，面向科研共同体，旨在推动小鼠胚胎干细胞及其衍生组织的可控遗传学研究。