Roukos Vassilis, Burgess Rebecca C, Misteli Tom
National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.
Nat Protoc. 2014 Oct;9(10):2476-92. doi: 10.1038/nprot.2014.167. Epub 2014 Sep 25.
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.
传统的DNA损伤生成方法不太适合观察受损染色体位点的时空特征。我们描述了一种细胞系统的衍生方案，用于在活细胞中诱导并可视化哺乳动物基因组特定位点的染色体损伤。该系统基于两侧带有细菌LacO/TetO操纵子阵列的核酸内切酶I-SceI识别位点的稳定整合，并结合逆转录病毒介导的荧光阻遏物（LacR/TetR）整合，以可视化LacO/TetO位点。I-SceI核酸内切酶的表达在整合位点特异性诱导双链断裂（DSB），并允许通过延时显微镜观察受损染色质的动态变化。在多条染色体中依次进行LacO-I-SceI/TetO-I-SceI整合，可生成一个用于可视化活细胞中染色体易位形成的系统。该方案需要中级细胞培养和分子生物学技能，并且适用于在约3-5个月内高效衍生出任何感兴趣的整合克隆报告系统。
