Enhanced green fluorescent protein as reporter protein for biomonitoring of cytotoxic effects in mammalian cells

Abstract

Biomonitoring of cytotoxic and genotoxic effects in mammals which allows fast and reliable quantification of these effects requires the use of an appropriate reporter in a mammalian cell system. Enhanced green fluorescent protein (EGFP) as an in vivo reporter is extremely useful for examining constitutive gene expression and the time scale of induction of a promoter in question by a variety of pollutants. For measurement of cytotoxic effects on mammalian cells, the EGFP gene under control of the CMV promoter (pEGFP-N1) was stably transfected into Chinese hamster ovary (CHO) cells and EGFP expressing clones were selected and expanded using the aminoglycoside antibiotic G418. Growth was determined by cell counts and measurements of fluorescence intensities in a microplate reader. Increase of fluorescence is delayed compared to the increase of cell numbers. After UVC-irradiation CHO-pEGFP-N1 cells show a dose-dependent prolonged lag phase and a lower maximal fluorescence. For measuring genotoxic effects, the EGFP gene has to be under control of a DNA damage inducible promoter. The resistance of EGFP to intracellular protease digestion may be useful for measuring long-lasting effects on gene expression. Kinetic measurements concerning the up and down regulation of gene expression can be performed with the destabilised d2EGFP, which has a half-life of 3 h in CHO cells. Due to decreased accumulation of the reporter protein the d2EGFP expressing cells emit lower absolute fluorescence. In consequence, the measurement of d2EGFP expression regulated by constitutive or inducible promoters should be achieved by FACS-analysis.