Determination of UV-induced DNA damages to suppress protein expression using reporter gene assay-based single cell cotransfection imaging cytometry.

摘要

Effects of UVB or UVC-induced DNA damage were simultaneously monitored at single cellular level by analyzing the change of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) expression in human embryonic kidney (HEK) 293 cells using multicolor single-cell imaging cytometry. The method is based on the idea that there exists a quantitative correlation between the degree of UV-induced DNA damage and protein expression. A cotransfection assay was performed using UVB irradiated YFP and UVC irradiated RFP genes to eliminate cell-to-cell variation in protein expression yield. Up to an UVB irradiation dose of 50kJ/m(2), YFP expression yield did not change compared to control. On the other hand, RFP expression yield decreased remarkably as the UVC dose increased from 79.5 to 159J/m(2). The results showed that a certain level of DNA damage is efficiently repaired by intracellular repair mechanism and does not influence protein mutation. In addition, it was found that the amount of DNA damage induced by UVB in sunlight would not interfere with normal protein expression in the human body. Single-cell imaging cytometry is a cell lysis-free approach to directly monitor the intracellular correlation between the degree of UV-induced DNA damage and protein expression.