SINGLE CELL REPORTER GENE ASSAY FOR HORMONE RECEPTORS

基因报告系统用于研究真核基因的表达和调控。广泛使用的细胞内报告基因无法分离具有确定转录反应的单个变体，因为它们需要在分析之前进行细胞裂解和提取。此外，这些测定缺乏单细胞分辨率。分泌型报告基因测定，例如SEAP（分泌型碱性磷酸酶）系统，提供了优于细胞内报告基因测定的改进，因为细胞裂解是不必要的。由于转染的细胞保持完整，基因表达的动力学可以很容易地研究使用相同的培养物，通过重复取样的媒体，减少测定时间和多个文化的变化。该I期SBIR提案旨在通过结合凝胶微滴（GMD）封装技术、新型分泌酶捕获形式、可沉淀的酶标记荧光底物和荧光激活细胞分选来开发灵敏、快速、高通量的SEAP报告基因测定。可以回收感兴趣的活的完整细胞用于随后的克隆。在I期研究中，将使用用于鉴定“孤儿”核激素受体激动剂的模型系统。拟议的商业应用：所提出的测定将提供用于筛选潜在治疗分子的化合物的大文库的快速高通量方法。据估计，全球治疗药物市场将达到1000亿美元。

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.