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Traditional methods for the generation of DNA damage are not well suited for the observation of spatiotemporal aspects of damaged chromosomal loci. We describe a protocol for the derivation of a cellular system to induce and to visualize chromosome damage at specific sites of the mammalian genome in living cells. The system is based on the stable integration of endonuclease I-SceI recognition sites flanked by bacterial LacO/TetO operator arrays, coupled with retroviral-mediated integration of their fluorescent repressors (LacR/TetR) to visualize the LacO/TetO sites. Expression of the I-SceI endonuclease induces double-strand breaks (DSBs) specifically at the sites of integration, and it permits the dynamics of damaged chromatin to be followed by time-lapse microscopy. Sequential LacO-I-SceI/TetO-I-SceI integrations in multiple chromosomes permit the generation of a system to visualize the formation of chromosome translocations in living cells. This protocol requires intermediate cell culture and molecular biology skills, and it is adaptable to the efficient derivation of any integrated clonal reporter system of interest in ≈ 3-5 months.

Generation of cell-based systems to visualize chromosome damage and translocations in living cells