GREEN FLUORESCENT PROTEIN (GFP) EXPRESSION IN MAMMALIAN CELLS AFTER UV-IRRADIATION

摘要

Green fluorescent protein (GFP) is a reporter system for monitoring in-vivo gene expression superior to other assays as GFP fluorescence does not require specific substrates or cofactors. CHO cells were co-transfected with the pCX-GFP ((3-actin/p-globin gene promoter) and a neomycin resistence transmitting plasmid. Resulting stable cell clones were selected and maintained using G418. CX-GFP codes for a red-shifted GFP-variant (488 nm excitation, 509 run emission) enabeling visualization of GFP expression by spectroscopy, fluorescence microscopy or FACS-analysis (488 nm excitation, FITC filters).rnGFP expressing cells were subjected to different fixation/staining protocols. Formaldehyde-fixed (FA) cells display GFP fluorescence comparable to living cells. Co-staining of DNA is possible in FA-fixed cells using Hoechst 33258 or DAPI. Propidium iodide (PI) staining of DNA is difficult, due to the fact that GFP-fluorescence in etha-nol-fixed samples is not stable and in FA-fixed samples there is some overlapping of emission spectra from GFP and PI.rnDNA repair proficient and deficient CHO strains were used for survival tests after UVC-irradiation. Cells carrying the GFP-construct show no difference in cellular survival compared to cells not carrying the construct.rnAfter certain post-irradiation growth periods cells were harvested and the numbers of GFP expressing cells and fluorescence were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (increasing GFP contents).