title: Generation of cell-based systems to visualize chromosome damage and translocations in living cells.
abstract: 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.
authors.affiliation: National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Roukos
authors.affiliation: National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Burgess
authors.affiliation: National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Misteli
mesh_terms: D000818:Animals; D002460:Cell Line; D002875:Chromosomes; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques / Q000295:instrumentation; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D008322:Mammals / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D009875:Operator Regions, Genetic; D010957:Plasmids; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods*; D014178:Translocation, Genetic
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific