Hb. Oral et al., A method for determining the cytoprotective effect of catalase in transiently transfected cell lines and in corneal tissue, ANALYT BIOC, 267(1), 1999, pp. 196-202
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.