ENZYME-GENERATED INTRACELLULAR FLUORESCENCE FOR SINGLE-CELL REPORTER GENE ANALYSIS UTILIZING ESCHERICHIA-COLI BETA-GLUCURONIDASE

We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus su bstrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammali an cells expressing and translating gus mRNA liberate sufficient level s of intracellular fluorescein for quantitative analysis by flow cytom etry. This assay can be used to FACS sort viable cells based on Gus en zymatic activity, and the efficacy of the assay can be measured indepe ndently by using a fluorometric lysate assay. Furthermore, both the be ta-glucuronidase and the previously described E. coli beta-galactosida se enzymes have high specificities for their cognate substrates, allow ing each reporter gene to be measured by FACS independently. (C) 1996 Wiley-Liss, Inc.