Visualization of the DNA repair process in mammalian cells transfected with EGFP-expressing plasmid DNA after exposure to X-rays in vitro
Visualization of the DNA repair process in mammalian cells transfected with EGFP-expressing plasmid DNA after exposure to X-rays in vitro
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体外暴露于 X 射线后，用表达 EGFP 的质粒 DNA 转染的哺乳动物细胞中 DNA 修复过程的可视化
DOI：
10.1093/rpd/ncy241
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发表时间：
2018
影响因子：
1
通讯作者：
Akinari Yokoya
中科院分区：
文献类型：
--
作者：
Hiroki Nakaue;Yui Obata;Kiichi Kaminaga;Nobuyoshi Akimitsu;Akinari Yokoya
为了研究电离辐射诱导的DNA损伤的修复过程，从不同类型的细胞质损伤中分离出来，我们使用脂质体将X射线照射的增强型绿色荧光蛋白（EGFP）表达质粒DNA转染到未照射的哺乳动物细胞中。在显微镜下观察细胞中照射质粒的修复动力学，作为转染细胞发射的EGFP荧光。使用琼脂糖凝胶电泳方法，还定量了质粒的单链和双链断裂的产率。作为阳性对照实验，还将具有由切口或限制性酶诱导的单链或双链断裂的质粒DNA转染到细胞中。X射线照射质粒的DNA修复率显著低于酶消化阳性对照样品。这些结果表明，X射线可以诱导较少的可修复的损伤比酶诱导。
To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.