Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish
Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish
复制标题
开发用于研究斑马鱼 DNA 双链断裂修复的新型 Visual-Plus 定量分析系统
DOI：
10.1016/j.jgg.2012.07.009
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发表时间：
2012-09-01
影响因子：
5.9
通讯作者：
Chen, Jun
中科院分区：
文献类型：
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作者：
Liu, Jingang;Gong, Lu;Chen, Jun
基于增强型绿色荧光蛋白（EGFP）和大范围核酸酶（如I-Sce I），使用报告系统分析DNA双链断裂（DSB）修复通常使用细胞系进行。在这项研究中，我们开发了三个视觉加定量分析系统的同源重组（HR），非同源末端连接（NHEJ）和单链退火（SSA）DSB修复途径在生物体水平上在斑马鱼胚胎。为了启动DNA DSB修复，我们使用了两个相反方向的I-Sce I识别位点，而不是通常的单个位点。三种I-Sce I-cut构建体分别启动了NHEJ、HR和SSA修复途径，并通过EGFP在胚胎中的表达追踪了I-Sce I引起的DNA损伤修复。除了监测绿色荧光强度外，还可以通过定量实时聚合酶链反应（qPCR）精确测量修复频率。对斑马鱼胚胎DSB位点DNA序列的分析表明，NHEJ在这三种修复途径中占主导地位。此外，虽然HR和SSA报告系统可以分别通过敲低rad 51和rad 52来有效地降低，但是当NHEJ构建体在体内被I-Sce I切割时，NHEJ只能通过敲低ligaseIV（lig 4）来受损。更有趣的是，用lig 4-MO阻断NHEJ增加了HR的频率，但降低了SSA的频率。我们的研究表明，用于修复DNA DSB的主要机制是保守的，从斑马鱼到哺乳动物，斑马鱼提供了一个很好的模型，研究和操纵DNA DSB修复在生物体水平。
The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.