The tumor suppressor Rb is functionally inactivated in most human, cancers and the consequent deregulation of E2F family members and their transcriptional targets is believed to be a crucial step in tumorigenesis. Based on their opposing transcriptional properties, the E2Fs can be divided into two classes: the activating E2Fs and the repressive E2Fs. There has been much debate about the relative roles of the activating versus repressive E2Fs in controlling the proliferation of both normal and tumor cells. Moreover, the role of these proteins in ES cell regulation has not yet been addressed. The goal of this proposal is to create a novel shRNA vector system that will enable inducible knockdown of multiple E2Fs in either cultured cells or mouse models. These studies will allow us to directly address the requirement of the activating E2Fs in ES cells, primary fibroblasts and in tumor development. Moreover, it will also provide a valuable vector resource to the community that can be used for efficient, simultaneous knockdown of genes of choice. ? ? ?