A Novel Approach for Real-Time Proteomics in Live Cells

DESCRIPTION (provided by applicant): The ultimate aim of this project is to develop a novel proteomic system that will enable the real-time expression profiling of proteins in live murine embryonic stem (mES) cells and, ultimately, in differentiated cells derived therefrom. In the first step, a library of clonal mES cell lines will be established, each of which will harbor a single fluorescently-tagged protein. The fluorescence in each cell of a particular clonal line will serve as a reporter for the expression level and subcellular localization of the tagged protein in that cell line. This library of clonal fluorescently-tagged lines can be subjected to quantitative fluorescence microscopy in multi- well live-cell arrays, to enable the simultaneous real-time expression profiling of multiple proteins in live cells under various culture conditions. This Phase I study will be focused primarily on developing as extensive a library in mES cells as is practically possible. Such a 'master' library of fluorescently-tagged mES cell lines can also be manipulated in culture into specific tissue-types such as cardiomyocytes, to create a powerful new system for biomarker discovery and drug-screening relevant to economically important diseases such as cardiovascular disease. Further, these mES cells can also be used for the generation of transgenic mice as and when desired, to extend such studies to live animals. The ultimate aim of this project is to develop a system that will enable researchers to monitor the location and levels of hundreds of proteins simultaneously in living cells. Such a system will be useful in understanding the molecular basis of a number of human diseases, such as cardiovascular disease, neurodegenerative disease and diabetes. Further, such a system can be applied to the development of novel diagnostic tests, identification therapeutic targets and the creation of powerful drug-screening platforms to identify potential therapeutic agents that may cure these diseases.