Green fluorescent protein (GFP) as a marker for cell viability after UV irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing gree n fluorescent protein (GFP) was achieved using a plasmid vector that encode d the red-shifted pCX-xGFP under the control of a strong hybrid promoter co mposed of a CMV enhancer and a beta-actin/ beta-globin,gene promoter. Cotra nsfection of the promoter-less pSV2-Neo helper plasmid transmitting neomyci n resistance was followed by selection with the antibiotic G418. Constituti ve GFP expression could be visualized in living and fixed cells using fluor escence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repa ir-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV S-pGFP) show the same response to UV irradiation (colony forming ability) a s their nontransformed parental cell lines (AA8, UV5). Using GFP as a marke r for cell viability, cells were harvested after certain postirradiation gr owth periods and the numbers of GFP expressing cells and fluorescence inten sities were determined by FAGS analysis. Generally, GFP fluorescence in irr adiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuo usly (leading to a dose-dependent increase in GFP contents).