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Both when developing gene constructs for therapeutic purposes and when testing the biological function of proteins, it would be convenient to use cells or tissues that have been transiently transfected with the gene of interest. However, determining the protective effects of transient gene expression is complicated by a low transfection efficiency, resulting in only a minority of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this study we have developed a convenient technique for determining cell death in transiently transfected vascular endothelial cell monolayers and in corneal tissue. Vascular endothelial cells were cotransfected with human catalase cDNA and the lacZ gene encoding beta-galactosidase, under conditions in which cells expressing beta-galactosidase also expressed catalase. By assaying release of beta-galactosidase upon cell death, it was possible to show that catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was adapted to demonstrate the protective effects of catalase transfection on hydrogen peroxide-mediated injury of intact corneal endothelium under ex vivo culture conditions. This assay should also be useful for characterizing the cytoprotective effects of other genes in transient transfection systems.

Type

Journal article

Journal

Analytical biochemistry

Publication Date

02/1999

Volume

267

Pages

196 - 202

Addresses

Division of Medicine, BHF Cardiovascular Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Du Cane Road, London, W12 ONN, United Kingdom.

Keywords

Endothelium, Vascular, Endothelium, Corneal, Cell Line, Hela Cells, CHO Cells, Animals, Humans, Hydrogen Peroxide, Catalase, DNA Primers, Transfection, Cell Death, Gene Expression, Base Sequence, Lac Operon, Cricetinae