Sensitive and rapid detection of beta-galactosidase expression in intact cells by microinjection of fluorescent substrate.
Bacterial beta-galactosidase, coded by lacZ, is a widely used reporter for studies of transcriptional activity of eukaryotic promoters at the single cell level. Unfortunately, current detection methods, like X-gal cytochemistry, are slow, have suboptimal sensitivity, and are incompatible with cell survival. By a novel approach based on microinjection into cells of the fluorogenic substrate 5-chloromethylfluorescein di-beta-D-galactopyranoside lacZ gene expression was detected without affecting cell viability or proliferative capacity. The method was far more sensitive than the conventional X-gal cytochemistry in all cell systems tested (primary hepatocytes, fibroblasts, and glioma cells). Results were obtained within seconds to minutes after injection, and cells remained fluorescent for hours.
Brustugun
O T
OT
University of Bergen, Department of Anatomy and Cell Biology, Norway.
Mellgren
G
G
Gjertsen
B T
BT
Bjerkvig
R
R
Døskeland
S O
SO
D000818:Animals; D001345:Autoradiography; D002461:Cell Line, Transformed; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005697:Galactosides; D006801:Humans; D007211:Indoles; D008845:Microinjections; D008856:Microscopy, Fluorescence; D051381:Rats; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005697:Galactosides; D007211:Indoles; C069306:5-chloromethylfluorescein; D001616:beta-Galactosidase; D016650:Fluorescein-5-isothiocyanate; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside