Figure 1: An RNAi screen for identifying repressors of germ cell-related gene expression in mouse ESCs.

(a) VR15, a mouse ESCs line that carries an RFP reporter driven by the Vasa promoter. In this schematic diagram, genes that repress germ-cell-related genes are inactivated by RNAi; consequently, the Vasa::RFP reporter is activated and easily monitored via fluorescence. (b) Top, a schematic diagram of the RNAi screen. The screen was performed using a 96-well format; siRNAs were transfected into VR15 with the reverse method (see Methods). Images of whole-well fluorescent in each well of the 96-well dishes were obtained daily. We tiled the images of 96 wells and searched for the wells that showed significantly brighter fluorescence than negative control wells. Bottom, magnified examples of images of an RFP-positive well and an RFP-negative well. Scale bar, 1 mm. (c) A tiled image that represents the time-dependent changes in RFP fluorescence following KD of each candidate gene in VR15 cells. (d) Phase contrast image (top) and an RFP fluorescence image (bottom) of Max-KD VR15 cell on day3. Scale bar, 100 μm. (e) Flow cytometry analysis of control VV3 (left) and Max-KD VV3 (right) cells. Robust emergence of a Vasa::Venus-positive cell population was observed in the Max-KD cultures.