2008-03-01
Induction of linear tracks of DNA double-strand breaks by α-particle irradiation of cells
Publication
Publication
Nature Methods , Volume 5 - Issue 3 p. 261- 266
Understanding how cells maintain genome integrity when challenged with DNA double-strand breaks (DSBs) is of major importance, particularly since the discovery of multiple links of DSBs with genome instability and cancer-predisposition disorders. Ionizing radiation is the agent of choice to produce DSBs in cells; however, targeting DSBs and monitoring changes in their position over time can be difficult. Here we describe a procedure for induction of easily recognizable linear arrays of DSBs in nuclei of adherent eukaryotic cells by exposing the cells to α particles from a small Americium source (Box 1). Each α particle traversing the cell nucleus induces a linear array of DSBs, typically 10-20 DSBs per 10 μm track length. Because α particles cannot penetrate cell-culture plastic or coverslips, it is necessary to irradiate cells through a Mylar membrane. We describe setup and irradiation procedures for two types of experiments: immunodetection of DSB response proteins in fixed cells grown in Mylar-bottom culture dishes (Option A) and detection of fluorescently labeled DSB-response proteins in living cells irradiated through a Mylar membrane placed on top of the cells (Option B). Using immunodetection, recruitment of repair proteins to individual DSB sites as early as 30 s after irradiation can be detected. Furthermore, combined with fluorescence live-cell microscopy of fluorescently tagged DSB-response proteins, this technique allows spatiotemporal analysis of the DSB repair response in living cells. Although the procedures might seem a bit intimidating, in our experience, once the source and the setup are ready, it is easy to obtain results. Because the live-cell procedure requires more hands-on experience, we recommend starting with the fixed-cell application.
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doi.org/10.1038/nmeth.f.206, hdl.handle.net/1765/30399 | |
Nature Methods | |
Organisation | Erasmus MC: University Medical Center Rotterdam |
Stap, J., Krawczyk, P., van Oven, C., Barendsen, G., Essers, J., Kanaar, R., & Aten, J. (2008). Induction of linear tracks of DNA double-strand breaks by α-particle irradiation of cells. Nature Methods, 5(3), 261–266. doi:10.1038/nmeth.f.206 |