Identification of regulators of exit from na?ve pluripotent state by enhancer trap integration site mapping

To identify regulators of exit from the na?ve pluripotent state we performed an enhancer trap screen under sensitized conditions. Briefly, haploid mouse ESCs with a Puromycin resistance gene under the control of Oct4 regulatory elements were mutagenized using viruses harboring enhancer trap constructs and subjected to differentiation in the presence of 3uM CHIR with concomitant selection for Oct4 expression. Selected cells were harvested after 16, 20, and 23 days and the extracted genomic DNA, together with genomic DNA of cells taken before selection, subjected to inverse PCR and sequencing. This allowed for mapping of enhancer trap integration sites and identifying genes whose targeting is enriched during selection.