NEW EGFP CALIBRATION STANDARDS FOR FLOW CYTOMETRY

适用于流式细胞术的新 EGFP 校准标准

• 批准号: 2874335
• 负责人: Abe Schwartz
• 金额: $ 6.24万
• 依托单位: CARIBBEAN MICROPARTICLES CORPORATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2874335
• 关键词: biotin; diagnosis quality /standard; flow cytometry; fluorescence; green fluorescent proteins; physical property; recombinant proteins; spectrometry; technology /technique development

The ability to clone green fluorescent protein(GFP) and express it in heterologous systems has resulted in a unique genetic reporter system. The development of GFP mutants further facilitated the study of gene expression and protein localization by flow cytometric analysis, confocal laser microscopy and fluorescence microscopy. The long-term goal of this proposal is to develop a set of GPF calibration standards that will allow investigators to quantify the number of GFP molecules expressed per cell. A mutant of GFP (EGFP) will be attached to the microbeads to obtain the necessary uniformity and spectral properties. Phase I studies will focus on producing microbead calibration standards using an EGFP biotinylated derivative; characterizing and assigning calibration values to these standards in Molecules of Equivalent Soluble Fluorochrome (MESF) units, and evaluating them in external laboratories. Research collaborations with Los Alamos National Laboratory and the UPR School of Medicine are expected to provide the technology transfer necessary to successfully develop the EGFP calibration standards. Quantification of GFP will provide new opportunities for innovative applications in gene expression analysis and will require products that will facilitate and ensure the precision and accuracy of quantitative measurements across GFP variants. PROPOSED COMMERCIAL APPLICATIONS: In the estimated 10,000 flow cytometry laboratories worldwide, increasing numbers of researchers are using GFP probes as research tools and will need to quantify the levels of expression. If GFP calibration standard kits sell for a price similar to those of other fluorescent probes and they are used with the same frequency (cost: $2,000/year) and only 25% of the laboratories become involved in GFP research, then it is estimated that the potential market would be $5,000,000 annually. Several companies have expressed already their interest in distributing GFP calibration standards.

克隆绿色荧光蛋白（GFP）并在异源系统中表达的能力形成了一个独特的遗传报告系统。GFP突变体的开发进一步促进了流式细胞分析、激光共聚焦显微镜和荧光显微镜对基因表达和蛋白定位的研究。该提案的长期目标是开发一套GFP校准标准，使研究人员能够量化每个细胞表达的GFP分子的数量。将GFP突变体（EGFP）附着在微珠上以获得必要的均匀性和光谱特性。第一阶段的研究将侧重于使用EGFP生物素化衍生物生产微珠校准标准；在等效可溶性荧光染料分子（MESF）单位中描述和分配这些标准的校准值，并在外部实验室对其进行评估。预计与洛斯阿拉莫斯国家实验室和普遍定期审议医学院的研究合作将提供成功开发EGFP校准标准所需的技术转让。GFP的量化将为基因表达分析的创新应用提供新的机会，并将需要能够促进和确保GFP变体定量测量的准确性和准确性的产品。建议的商业应用：在全球估计有10,000个流式细胞术实验室中，越来越多的研究人员正在使用GFP探针作为研究工具，并且需要量化表达水平。如果绿色荧光蛋白校准标准试剂盒的售价与其他荧光探针相似，并且使用频率相同（成本：2000美元/年），并且只有25%的实验室参与绿色荧光蛋白研究，那么估计潜在市场将为每年500万美元。一些公司已经表达了他们对分发绿色荧光蛋白校准标准的兴趣。