Creation of a Versatile Proteomic Resource in Murine Embryonic Stem Cells

DESCRIPTION provided by applicant The ultimate goal of this project is to create a novel proteomic resource that will enable the real time expression profiling of proteins in live murine embryonic stem mES cells and in differentiated cells derived therefrom The resource will be in the form of an annotated bank of protein trap mES cell lines each of which will carry a single tagged gene Fluorescent signal in these lines will report the expression and subcellular localization of the protein product s of the tagged gene in each line Such a library of clonal fluorescently tagged lines can be used in conjunction with quantitative fluorescence microscopy in multiwell live cell arrays to study thousands of proteins in live cells in real tim under various experimental conditions In a separate effort we have initiated the development of such a bank of fluorescent protein trap mES cell lines However the technology we have used so far will only allow us to detect and isolate protein traps in genes that are actively expressed in mES cells this limits the breadth and versatility of the library because the bulk of the proteome andgt is estimated to be effectively silent in mES cells In order to create a truly broad proteomic library we have devised a method that will allow us to identify and isolate protein traps in genes that are silent in mES cells Further in this new approach we will also add a more versatile protein tag FAP tag so as to greatly increase the sensitivity of fluorescence detection in and therefore the overall functionality of the protein trap mES library This Phase I project is designed to construct the requisite DNA vectors to test their efficacy at identifying protein traps in silent genes and to assess their scope and suitability for use in a future large scale protein trapping effort PUBLIC HEALTH RELEVANCE The ultimate aim of this project is to develop a large and versatile proteomic resource that will enable researchers to study thousands of proteins simultaneously in living cells and in real time This proteomic resource will enhance a wide range of basic and applied research On the one hand it will enhance research in the understanding of the molecular basis of many human diseases On the other hand it will also enable the development of novel in vivo assays such as drug screening and toxicity screening assays that will accelerate the discovery and development of more powerful and safer therapeutic agents to treat human diseases