Exploration of self-renewal and pluripotency in ES cells using RNAi

Embryonic stem cells (ESCs) have the ability to expand indefinitely in vitro and give rise to cells of all three germ layers as well as germ cells. For these reasons, ESCs hold great promise for biomedicine. In order to harness the potential of pluripotent cells, it is necessary to first understand the molecular mechanisms that control the pluripotent state. The discovery of RNA interference has made such functional analysis, even at high(er) throughput, possible. Here, we describe the methods used for high-throughput siRNA screening by high-content microscopy to identify gene products that regulate mouse ESC fate decision. In addition, we will describe the application of lentivirus-based shRNA knockdown to explore or validate the role of candidate genes in ESC pluripotency.