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 substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. Furthermore, both the beta-glucuronidase and the previously described E. coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently.
Lorincz
M
M
Department of Genetics, Stanford University Medical School, California, USA.
Roederer
M
M
Diwu
Z
Z
Herzenberg
L A
LA
Nolan
G P
GP
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000032:analysis; D017930:Genes, Reporter; D005937:Glucaric Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007202:Indicators and Reagents / Q000032:analysis; D051379:Mice; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism
D017931:DNA Primers; D005452:Fluoresceins; D007202:Indicators and Reagents; D012333:RNA, Messenger; C041357:fluorescein glucuronide; C009194:saccharolactone; D001616:beta-Galactosidase; D005966:Glucuronidase; D005937:Glucaric Acid