shRNAi induced differentiation time courses in mouse embrionic stem cells.

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem (ES) cells. Short hairpin RNA (shRNA) techniques are employed to down regulate a set of gene-products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven molecules whose shRNA-mediated depletion induces differentiation, including four whose roles in self-renewal had not been demonstrated. We analyze the expression profiles of embryonic stems (ES) cells transduced with individual shRNA vectors, maintained in the presence of LIF. Keywords: time course We analyze transcriptome dynamics after down-regulating each of the 8 self-renewal regulators identified in the current studies. These are Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1, Mm.343880 and Dppa4. Gene specific shRNA transduced GFP+ cells were FACS purified daily during a seven day interval and used to interrogate Affymetrix microarrays. Empty H1P vector served as a reference.