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.
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Acknowledgements
Work in the Misteli laboratory is supported by the Intramural Research Program of the US National Institutes of Health (NIH), National Cancer Insitute (NCI), Center for Cancer Research. We thank E. Soutoglou and C. Schmidt for the optimization of specific steps of this protocol, and we thank T. Karpova (NCI Fluorescence Imaging Microscopy Facility) for help with microscopy.
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V.R. developed the protocol. V.R. and R.C.B. wrote the manuscript. T.M. supervised the study and edited the paper.
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Integrated supplementary information
Supplementary Figure 1 Optimal size and density of clones.
Representative images of cell colonies that are: (a) smaller than optimal, (b) optimal or (c) colonies plated at a density higher than the desired.
Supplementary Figure 2 Retroviral construct maps and optimal plating density for Plat-GP cells.
A. Retroviral bicistronic vectors designed to express a fusion of Tet repressor (TetR) tagged with the fluorescent protein mCherry along with the puromycin selection marker (a) or the Lac repressor (LacR) fused to GFP along with a neomycin resistance gene (b) via an internal ribosome entry site (IRES) B. Plating density of Platinum-GP cells. Representative images of cells plated at confluencies that are: (a) lower than optimal, (b) optimal for transfection and (c) overconfluent.
Supplementary Figure 3 Cells with high expression of the fluorescent repressors.
High expression of the fluorescent repressors prevents the visualization of LacO/TetO arrays (arrows). Nuclei stained with Hoechst. Scale bar is 10 μm.
Supplementary information
Supplementary Figure 1
Optimal size and density of clones. (PDF 993 kb)
Supplementary Figure 2
Retroviral construct maps and optimal plating density for Plat-GP cells. (PDF 1256 kb)
Supplementary Figure 3
Cells with high expression of the fluorescent repressors. (PDF 1575 kb)
Supplementary Table 1
A list of the plasmids used in this protocol. (PDF 75 kb)
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Roukos, V., Burgess, R. & Misteli, T. Generation of cell-based systems to visualize chromosome damage and translocations in living cells. Nat Protoc 9, 2476–2492 (2014). https://doi.org/10.1038/nprot.2014.167
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DOI: https://doi.org/10.1038/nprot.2014.167
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