SINGLE CELL REPORTER GENE ASSAY FOR HORMONE RECEPTORS

激素受体单细胞报告基因测定

• 批准号: 6071693
• 负责人: PHILLIP T MOEN
• 金额: $ 9.52万
• 依托单位: ONE CELL SYSTEMS, INC
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6071693
• 关键词: alkaline phosphatase; enzyme substrate; flow cytometry; fluorescent dye /probe; gel; high throughput technology; hormone receptor; microcapsule; molecular cloning; reporter genes; technology /technique development; tissue /cell culture; transfection

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.

基因报告系统被用来研究真核基因的表达和调控。广泛使用的细胞内报告不能分离出具有明确转录反应的单个变体，因为它们在分析之前需要细胞裂解和提取。此外，这些分析缺乏单细胞分辨率。分泌性报告分析，如分泌性碱性磷酸酶(SEAP)系统，提供了比细胞内报告分析更好的方法，因为细胞裂解是不必要的。由于转基因细胞保持完好，通过重复采样介质，可以很容易地使用相同的培养物来研究基因表达的动力学，从而减少分析时间和多个培养物的变异性。这一阶段的SBIR方案旨在结合凝胶微滴(GMD)封装技术、新的分泌酶捕获形式、可沉淀的酶标记荧光底物和荧光激活细胞分选技术，开发一种灵敏、快速、高通量的SEAP报告基因分析方法。活的、完整的感兴趣细胞可以被回收用于随后的克隆。在第一阶段的研究中，将使用一个模型系统来识别“孤儿”核激素受体的激动剂。建议的商业应用：建议的测试将提供一种快速、高通量的方法，用于筛选潜在治疗分子的大型化合物文库。据估计，治疗技术在全球是一个1000亿美元的市场。