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.
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.
