携带HR-GFP报告基因的DNA损伤同源重组修复检测系统的建立及初步应用  

Establishment ofⅠ-Sce I-HR system with HR-GFP reporter gene and its preliminary application

作　　者：邓昭敏 李莎[1] 李明远[1] 王欢[1] 任来峰[1] 郭连娣 刘聪[2] 丁娜娜[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,四川成都610041 [2]四川大学华西第二医院发育与干细胞研究所,四川成都610041

出　　处：《西部医学》2013年第12期1770-1774,共5页Medical Journal of West China

基　　金：国家自然科学基金(81071362;30970950);中央高校基本科研业务基金(2010SCV21004)

摘　　要：目的建立DNA损伤同源重组修复检测系统(Ⅰ-Sce I-HR),应用该系统制备DNA双链断裂(DSB)的人骨肉瘤细胞(U2OS)模型,探索细胞DNA损伤后的修复特性奠定基础。方法通过分子克隆构建携带Ⅰ-Sce I归位内切酶识别序列的真核表达载体pcDNA3-HR-GFP,将该质粒转染入U2OS细胞中,经G418稳定筛选。随后分别瞬时转染入携带归位内切酶Ⅰ-Sce I的表达质粒pCBASCEI,以及体外经Ⅰ-Sce I线性化pcDNA3-HR-GFP。48小时后用免疫荧光方法检测DNA双链损伤效应分子-γ-H2AX,同时观察EGFP荧光信号;72小时后用Western blot检测报告蛋白EGFP的表达,评估DNA双链断裂后同源重组修复情况。结果酶切鉴定和测序证实pcDNA3-HR-GFP真核表达载体构建成功;Ⅰ-Sce I-HR系统引入U2OS细胞中后,γ-H2AX表达明显上调,荧光显微镜和Western blot均显示EGFP表达。结论 DSB细胞同源重组修复模型构建成功,Ⅰ-Sce I-HR系统能够成功地诱导U2OS细胞株产生DSB,并出现同源重组修复,为进一步研究DNA同源重组信号传导提供了有效的研究工具。Objective To construct the system of I-SceI-HR and induce a site-specific DNA double strand breaks （DSB）in human osteosarcoma cell line, U2OS and explore the properties of DNA repair using this system. Methods The eukaryotic expression plasmid pcDNA3-HR-GFP was constructed and transfected into U2OS cells. The positive neomy- cin-resistant transfeeted cell clones were generated by G418 selection. The positive cells containing the I -SceI endonucle- ase site were transfected instantaneously with the I -SceI expression plasmid, pCBASCE, as well as transfected with pcD- NA3-HR-GFP digested by I-Sce I in vitro separately. At 48h post-transfection, an early cellular marker of DSB,γ-H2AX, was detected using immunocytochemistry in both cells. The expression of EGFP was analyzed by fluorescent mi- croscope at 48h and by Western blot at 72h to represent homologous recombination （HR）. Results Restriction endonu- clease analysis and DNA sequencing confirmed that the plasmid pcDNA3-HR-GFP was successfully constructed. γ- H2AXs were induced in both cells, and EGFP expression increased significantly in cells transfected with I -SceI -HR system. Conclusion Genomie DSB could be induced in U2OS by introducing the I -SeeI -HR system,as well as homolo- gous recombination （HR） also happened. The cell model could be an effective tool for further study signal transduction in HR.

关 键 词：同源重组 DNA双链断裂 Ⅰ-Sce I-HR 

分 类 号：R34[医药卫生—基础医学] Q754[生物学—分子生物学]