A method for determining the cytoprotective effect of catalase in transiently transfected cell lines and in corneal tissue

Both when developing gene constructs for therapeutic purposes and when test ing the biological function of proteins, it would be convenient to use cell s or tissues that have been transiently transfected with the gene of intere st. However, determining the protective effects of transient gene expressio n is complicated by a low transfection efficiency, resulting in only a mino rity of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this st udy 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 whi ch cells expressing beta-galactosidase also expressed catalase, By assaying release of beta-galactosidase upon cell death, it was possible to show tha t catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was ad apted to demonstrate the protective effects of catalase transfection on hyd rogen 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, ( C) 1999 Academic Press.