An inducible and reversible embryonic stem cell biobank reveals functional genomic pathways and disease targets [iPCR/BC-PCR]. Mus musculus

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 Please note that [1] this study is used to create a biobank of clones which have a specific insertion and are uniquely identifieable. Due to the huge amount of clones the processed data would be very large and difficult to interpret without the context of the database. Therefore, the processed data will be made accessible to everyone at https://www.haplobank.at (upon acceptance of the manuscript) [2] the barcodes are used to identify the clones/wells in "4D" which are rows/columns/slices/towers [3] The protocols used for the samples are found in great detail at https://www.haplobank.at/ecommerce/control/haplobank_resource