Visualization of Heavy Ion Tracks by Labeling 3′-OH Termini of Induced DNA Strand Breaks
Visualization of Heavy Ion Tracks by Labeling 3′-OH Termini of Induced DNA Strand Breaks
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DOI：
10.1269/jrr.10097
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发表时间：
2011-07-01
影响因子：
2
通讯作者：
Hieda, Kotaro
中科院分区：
文献类型：
--
作者：
Konishi, Teruaki;Takeyasu, Akihiro;Hieda, Kotaro
用碳离子（LET：735 keV/μ m）和氩离子（LET：3，000 keV/μ m）照射非洲绿色猴肾细胞CV-1，通过标记DNA链断裂的3 ′-OH末端结果，观察穿过细胞核的离子轨迹。用TdT催化BrdU-三磷酸标记DNA的3 ′-OH末端。这种TUNEL（TdT介导的dUTP缺口末端标记）方法是基于TdT与DNA的3 '-OH末端的特异性结合。随后用抗BrdU的一级单克隆抗体进行免疫荧光染色，然后用Alexa Fluor 488的二级抗体进行免疫荧光染色，以使BrdU标记的DNA末端可视化。通过共聚焦激光显微镜获得细胞核的图像。当用氩离子垂直照射细胞单层时，在细胞核中诱导的DSB可被识别为荧光斑点。在另一个照射装置中，当用入射氩离子以小角度照射细胞时，DNA链断裂被检测为穿过细胞核的荧光条纹。这些结果证明了在DNA链断裂位点处的3 '-OH末端的诱导沿着氩离子轨迹。
African green monkey kidney cells, CV-1, were irradiated with Carbon ions (LET: 735 keV/mu m Argon ions (LET: 3,000 keV/mu m) to visualize ion tracks through the cell nucleus by labeling the 3'-OH termini result of DNA strand breaks. The 3'-OH termini of DNA were labeled with BrdU-triphosphate catalyzed by TdT. This method of TUNEL (TdT-mediated dUTP Nick End labeling) is based on the specific binding of TdT to 3'-OH termini of DNA. Subsequent immuno-fluorescent staining with the primary monoclonal antibody against BrdU, followed by a secondary antibody of Alexa Fluor 488, was performed to visualize the BrdU labeled DNA termini. Images of the cell nuclei were acquired by confocal laser microscopy. When cell monolayers were irradiated perpendicularly with argon ions, induced DSBs in cell nuclei were identifiable as fluorescent spots. In another irradiation setup, when cells were irradiated at a small angle with incident argon ions, DNA strand breaks were detected as fluorescent stripes across the cell nucleus. These results demonstrate the induction of 3'-OH termini at sites of DNA strand breaks along Argon ion tracks.