Induction of linear tracks of DNA double-strand breaks by α-particle irradiation of cells
Induction of linear tracks of DNA double-strand breaks by α-particle irradiation of cells
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DOI：
10.1038/nmeth.f.206
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发表时间：
2008-03
期刊：
影响因子：
48
通讯作者：
J. Stap;P. Krawczyk;C. Oven;G. Barendsen;J. Essers;R. Kanaar;J. Aten
中科院分区：
文献类型：
--
作者：
J. Stap;P. Krawczyk;C. Oven;G. Barendsen;J. Essers;R. Kanaar;J. Aten
了解细胞在面临 DNA 双链断裂 (DSB) 挑战时如何维持基因组完整性非常重要，特别是自从发现 DSB 与基因组不稳定和癌症易感性疾病的多重联系以来。电离辐射是在细胞中产生 DSB 的首选方法；然而，针对 DSB 并监测其地位随时间的变化可能很困难。在这里，我们描述了一种通过将细胞暴露于来自小镅源的 α 粒子（框 1）来在贴壁真核细胞的细胞核中诱导易于识别的 DSB 线性阵列的程序。每个穿过细胞核的 α 粒子都会产生 DSB 线性阵列，通常每 10 μm 轨道长度有 10-20 个 DSB。由于 α 粒子无法穿透细胞培养塑料或盖玻片，因此需要通过聚酯薄膜照射细胞。我们描述了两种类型实验的设置和照射程序：对在聚酯薄膜底部培养皿中生长的固定细胞中的 DSB 反应蛋白进行免疫检测（选项 A），以及在通过置于细胞顶部的聚酯薄膜照射的活细胞中检测荧光标记的 DSB 反应蛋白（选项 B）。使用免疫检测，最早在照射后 30 秒就可以检测到修复蛋白向各个 DSB 位点的募集。此外，与荧光标记的 DSB 响应蛋白的荧光活细胞显微镜相结合，该技术可以对活细胞中的 DSB 修复响应进行时空分析。尽管这些过程可能看起来有点令人生畏，但根据我们的经验，一旦源和设置准备就绪，就很容易获得结果。由于活细胞程序需要更多的实践经验，我们建议从固定细胞应用开始。
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.