A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase.
Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.
Lim
K
K
University of North Carolina.
Chae
C B
CB
D000818:Animals; D002478:Cells, Cultured; D004926:Escherichia coli; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005696:Galactosidases / Q000235:genetics*; D005819:Genetic Markers; D005822:Genetic Vectors; D051379:Mice; D008809:Mice, Inbred C3H; D009598:Nitrophenylgalactosides; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
D005819:Genetic Markers; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D005696:Galactosidases; D001616:beta-Galactosidase