Quantification of beta-galactosidase activity after non-viral transfection in vivo.
The limited efficacy of non-viral gene delivery systems currently hampers their wider therapeutic use. In order to further develop novel gene delivery systems, it is important to quantify their efficacy. Many reporter gene assays have limitations when being used to quantify expression in vivo. We have developed a simple assay which allows the quantification of beta-galactosidase transgene activity in vivo. The assay is based on beta-galactosidase cleavage of the DDAO-galactopyranoside substrate to DDAO, which shifts the fluorescence towards longer wavelengths. Reaction conditions were optimised to minimise degradation, activity of endogenous beta-galactosidase, and non-specific background fluorescence. The spectrofluorimetric quantification of the reaction product DDAO in the red part of the spectrum avoided interference from haemoglobin or other bio-molecules which hamper many in vivo assays. Routinely, amounts of less than 1 ng of beta-galactosidase (1 mU) per gram tissue could be detected and quantified. After intravenous administration of beta-galactosidase complexed with linear polyethylenimine (PEI, 22 kD) in mice, 134 mU g(-1) beta-galactosidase were detected in the lung, but only 2.9 mU g(-1) were found in the liver.
Zinselmeyer
Bernd H
BH
Cancer Research, UK Department of Medical Oncology, Beatson Laboratories, University of Glasgow, G61 1BD, Glasgow, UK.
Beggbie
Nicola
N
Uchegbu
Ijeoma F
IF
Schätzlein
Andreas G
AG
D000818:Animals; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics*
D006454:Hemoglobins; D001616:beta-Galactosidase