Differentiation stage-specific analysis of gene function with inducible short hair-pin RNA in differentiating embryonic stem cells

Cell differentiation is regulated by spatial and temporal coordination of gene expressions. Previously, we have established an embryonic stem (ES) cell differentiation system that can trace early cardiovascular developmental process in vitro. Here we show that tetracycline-induced short hair-pin RNA (shRNA) expression in differentiating ES cells successfully suppressed stage-specific genes for differentiation and modified cell fates. We established ES cell lines carrying shRNA gene driven by tRNAval promoter with tetracycline operator sequences (tet-ON system). When expression of vascular endothelial growth factor receptor-2 (VEGFR2) gene, a vascular progenitor and mesoderm marker and an essential gene for endothelial cell (EC) differentiation, was suppressed by shRNA in early ES cell differentiation, appearance of VEGFR2+ mesoderm cells was substantially reduced. Suppression of VEGFR2 expression at mesoderm stage almost completely inhibited EC differentiation from VEGFR2+ mesoderm cells. This novel experimental system, thus, can selectively determine stage-specific roles of genes in differentiation in vitro.