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
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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 ofⅠ-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 neomycin-resistant transfected cell clones were generated by G418selection.The positive cells containing theⅠ-SceI endonuclease site were transfected instantaneously with theⅠ-SceI expression plasmid,pCBASCE,as well as transfected with pcDNA3-HR-GFP digested byⅠ-Sce I in vitro separately.At 48hpost-transfection,an early cellular marker of DSB,γ-H2AX,was detected using immunocytochemistry in both cells.The expression of EGFP was analyzed by fluorescent microscope at 48hand by Western blot at 72hto represent homologous recombination(HR).Results Restriction endonuclease 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 Genomic DSB could be induced in U2OS by introducing theⅠ-SceI-HR system,as well as homologous recombination(HR)also happened.The cell model could be an effective tool for further study signal transduction in HR.
Objective To construct the system ofⅠ-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 neomycin-resistant transfected cell clones were generated by G418selection.The positive cells containing theⅠ-SceI endonuclease site were transfected instantaneously with theⅠ-SceI expression plasmid,pCBASCE,as well as transfected with pcDNA3-HR-GFP digested byⅠ-Sce I in vitro separately.At 48hpost-transfection,an early cellular marker of DSB,γ-H2AX,was detected using immunocytochemistry in both cells.The expression of EGFP was analyzed by fluorescent microscope at 48hand by Western blot at 72hto represent homologous recombination(HR).Results Restriction endonuclease 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 Genomic DSB could be induced in U2OS by introducing theⅠ-SceI-HR system,as well as homologous recombination(HR)also happened.The cell model could be an effective tool for further study signal transduction in HR.
引文
[1]Hava SR,Gilad M,Keren BB,et al.ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair[J].EMBO reports,2011,96(12):713-719.
[2]Galanty Y,Belotserkovskaya R,Julia C,et al.RNF4,a SUMO-targeted ubiquitin E3ligase[J].promotes DNA doublestrand break repair,2012,26(10):1196-1208.
[3]Roos WP,Nikolova T,Quiros S,et al.Brca2/Xrcc2dependent HR,but not NHEJ,is required for protection against O(6)-methylguanine triggered apoptosis,DSBs and chromosomal aberrations by aprocess leading to SCEs[J].DNA Repair,2009,8(1):72-86.
[4]Nakanishi K,Yang YG,Pierce AJ,et al.Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.Proc.Natl[J].Acad Sci USA,2005,102(5):1110-1115.
[5]任精华,何文山,林菊生,等.I-Sce I系统的建立及其在诱导肝癌细胞DNA双链断裂中的应用[J].中华肝脏病杂,2008,16(2):101-104.
[6]Weinstock DM,Jasin M.Alternative pathways for the repair of RAG-induced DNA breaks[J].Mol Cel Biol,2005,26(3):131-139.
[7]Zaunbrechera GM,Bashir M,Dunnea PW,et al.Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination(HR)to NonHomologous End Joining(NHEJ)[J].Animal Biotechnology,2008,19(1),6-21.
[8]Yang S,Chintapalli J,Sodagum L,et al.Activated IGF‐1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination[J].Am J Physiol Renal Physiol,2005,289(10):1144-1152.
[9]Akyuz N,Gisa S,Susse BS,et al.DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair[J].Molecular And Cellular Biology,2002,22(1):6306-6317.
[10]Lieber MR,Ma Y,Pannicke U,et al.Mechanism and regulation of human non-homologous DNA end-joining[J].Nat Rev Mol Cell Biol,2003,4(18):712-720.
[11]Myllynena L,Rieckmanna T,Jochen DDaphic,et al.In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53and K-Ras status[J].Radiotherapy and Oncology,2011,101(1):147-151.ed cell death and genotoxicity:Cell cycle dependence and the role of DNA double-strand breaks,HR and NHEJ.Cell Cycle[J].2012,11(14):2606-2619.
[12]Nikolova T,Hennekes F,Bhatti A,et al.Chloroethylnitrosoureainduced cell death and the role of DNA double-strand breaks,HR and NHEJ[J].Cell Cycle,2012,11(14):2606-2619.
[2]Galanty Y,Belotserkovskaya R,Julia C,et al.RNF4,a SUMO-targeted ubiquitin E3ligase[J].promotes DNA doublestrand break repair,2012,26(10):1196-1208.
[3]Roos WP,Nikolova T,Quiros S,et al.Brca2/Xrcc2dependent HR,but not NHEJ,is required for protection against O(6)-methylguanine triggered apoptosis,DSBs and chromosomal aberrations by aprocess leading to SCEs[J].DNA Repair,2009,8(1):72-86.
[4]Nakanishi K,Yang YG,Pierce AJ,et al.Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.Proc.Natl[J].Acad Sci USA,2005,102(5):1110-1115.
[5]任精华,何文山,林菊生,等.I-Sce I系统的建立及其在诱导肝癌细胞DNA双链断裂中的应用[J].中华肝脏病杂,2008,16(2):101-104.
[6]Weinstock DM,Jasin M.Alternative pathways for the repair of RAG-induced DNA breaks[J].Mol Cel Biol,2005,26(3):131-139.
[7]Zaunbrechera GM,Bashir M,Dunnea PW,et al.Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination(HR)to NonHomologous End Joining(NHEJ)[J].Animal Biotechnology,2008,19(1),6-21.
[8]Yang S,Chintapalli J,Sodagum L,et al.Activated IGF‐1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination[J].Am J Physiol Renal Physiol,2005,289(10):1144-1152.
[9]Akyuz N,Gisa S,Susse BS,et al.DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair[J].Molecular And Cellular Biology,2002,22(1):6306-6317.
[10]Lieber MR,Ma Y,Pannicke U,et al.Mechanism and regulation of human non-homologous DNA end-joining[J].Nat Rev Mol Cell Biol,2003,4(18):712-720.
[11]Myllynena L,Rieckmanna T,Jochen DDaphic,et al.In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53and K-Ras status[J].Radiotherapy and Oncology,2011,101(1):147-151.ed cell death and genotoxicity:Cell cycle dependence and the role of DNA double-strand breaks,HR and NHEJ.Cell Cycle[J].2012,11(14):2606-2619.
[12]Nikolova T,Hennekes F,Bhatti A,et al.Chloroethylnitrosoureainduced cell death and the role of DNA double-strand breaks,HR and NHEJ[J].Cell Cycle,2012,11(14):2606-2619.