Live-cell visualisation of chromatin for the study of radiation-induced chromosome aberrations

Abstract

Chromosomes can be visualised during the metaphase stage of the cell cycle however, there are various limitations associated with the visualisation of chromatin during interphase. This is due to the fact that during that stage of the cell cycle, chromatin is decondensed and difficult to resolve by using standard techniques. The employment of fluorescence microscopy has provided great insight into the examination of the structure and dynamics of fluorescently tagged chromatin regions in living cells during interphase. Thus the in vivo stable expression of fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) has the potential to address questions that lead to understanding dynamic responses of chromatin in response to DNA damage. In this study, chromatin sites within the nuclei of Chinese Hamster Ovary (CHO) cells were fluorescently labelled using the lac operon system, which works on the principle that EGFP-lac repressor protein will bind to sites of integrated lac operator sequence. To achieve this, CHO cells were transfected with pSV2-DHFR8.32 (lac operator) and p3‟SSdimerClonEGFP (lac repressor-EGFP fusion protein) plasmids by calcium phosphate transfection. 68 stable transfectants were stored and clone CHO-R-O-25 cells were used for further experimental analysis based on these cells containing discrete and intense fluorescent spots. It was shown that the physiological and functional characteristics of CHO-R-O-25 cells were comparable to those of control CHO DG44 cells. Time-lapse fluorescence microscopy was used to examine for any alterations in the physical structure of chromatin in response to DNA damage that was induced by radiation and a range of different chemicals. It was demonstrated that in cells exposed to DNA damaging agents, chromatin diffusion and expansions revealed by alterations in the intensity and size of the fluorescent spots within the nuclei of cells are owed to relaxation of chromatin structures in response to genome insults. The potential for this tool to study the organisational responses of chromatin in response to damage and the ulitimate relevance of any physical alterations to mechanisms for chromosome exchange formation, are described.