SINGLE CELL REPORTER GENE ASSAY FOR HORMONE RECEPTORS

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Genetic reporter systems are used to study eukaryotic gene expression and regulation. Widely used intracellular reporters cannot isolate individual variants with defined transcription responses because they require cell lysis and extraction prior to analysis. Furthermore, these assays lack single cell resolution. Secreted reporter assays, such as the SEAP (secreted alkaline phosphatase) system, offer an improvement over intracellular reporter assays because cell lysis is unnecessary. Since the transfected cells remain intact, the kinetics of gene expression can be easily studied using the same cultures by repeatedly sampling the media, reducing assay time and multiple culture variability. This Phase I SBIR proposal is aimed at developing a sensitive, rapid, high throughput SEAP reporter gene assay by combining gel microdrop (GMD) encapsulation technology, a novel secreted enzyme capture format, a precipitable, enzyme-labeled fluorogenic substrate, and fluorescence activated cell sorting. Live, intact cells of interest can be recovered for subsequent cloning. In Phase I studies, a model system for identifying agonists to "orphan" nuclear hormone receptors will be used. PROPOSED COMMERCIAL APPLICATIONS: The proposed assay will provide a rapid high throughput method for screening large libraries of compounds for potential therapeutic molecules. Therapeutics is estimated to be a $100 billion market worldwide.