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

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. Overall design: Genomic integration sites of mutagens were mapped by inverse PCR https://www.haplobank.at/ecommerce/control/haplobank_resource