Developing shRNA vectors to study the activating E2Fs

描述（由申请人提供）：肿瘤抑制因子Rb在大多数人类癌症中是功能失活的，因此E2F家族成员及其转录靶点的失调被认为是肿瘤发生中的关键步骤。根据其相反的转录特性，E2Fs可以分为两类：激活型E2Fs和抑制型E2Fs。关于激活型E2Fs与抑制型E2Fs在控制正常细胞和肿瘤细胞增殖中的相对作用存在很多争论。此外，这些蛋白质在ES细胞调控中的作用尚未得到解决。该提案的目标是创建一种新的shRNA载体系统，该系统将能够在培养细胞或小鼠模型中诱导敲低多个E2F。这些研究将使我们能够直接解决ES细胞，原代成纤维细胞和肿瘤发展中激活E2Fs的需求。此外，它还将为社区提供有价值的载体资源，可用于有效地同时敲除选择的基因。

DESCRIPTION (provided by applicant): 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.