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Homologous Recombination Assay
同源重组分析   

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Abstract

Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme.

Keywords: DNA double strand break repair(DNA双链断裂修复), Homologous recombination(同源重组), Human cells(人体细胞), DNA repair(DNA修复), Genetic instability(遗传不稳定性)

Materials and Reagents

  1. U2OS or RG37 cell lines
    Note: U2OS are osteosarcoma cells with active p53 pathway, RG37 are SV40-immortalized human fibroblasts with inactive p53 pathway. There are cells harbouring the substrate designed to measure Homology Directed repair. However, any cell line in which the HDR substrate has been inserted can be used.
  2. Culture medium (serum-free medium)
  3. siRNA for gene of interest (for example rad51, its depletion decreases the efficiency of repair of a DNA DSB by homologous recombination)
  4. Plasmid pcDNA3myc-NLS-I-SceI (coding for I-SceI created by Puget et al. (2005))
  5. INTERFERin Transfection Reagent (Ozyme, catalog number: POL409-10 )
  6. JetPEI Transfection Reagent (Ozyme, catalog number: POL101-10 )
  7. Trypsin
  8. Glycerol
  9. SDS
  10. Beta-mercaptoethanol
  11. Bromophenol blue
  12. Laemmli buffer (see Recipes)
  13. Phosphate Buffered Saline (PBS) (see Recipes)

Equipment

  1. 35 mm tissue culture plate
  2. Tissue culture set up (e.g. 37 °C, 5% CO2 incubator)
  3. Flow cytometer
  4. Western blotting apparatus

Procedure

  1. Plate 100,000 cells (U2OS or RG37) per 35 mm diameter plate at the end of the afternoon. Cells are cultured in humidified atmosphere in a cell incubator at 37 °C with 5% CO2.
  2. Let attach overnight.
  3. Transfect with siRNA using INTERFERin according to the manufacturer’s instructions. Use 10 nM of siRNA mixed in 200 μl of serum-free medium with 8 μl of INTERFERin.
  4. 24 h later, change the medium and transfect with the plasmid coding for I-SceI in order to induce DNA double strand breaks in the GFP copy harboring the I-SceI recognition site. Use 1 μg of plasmid per plate. Transfect using JetPEI according to the manufacturer’s instructions by mixing 1 μg of I-SceI plasmid in 200 μl of NaCl (150 mM) with 2 μl of JetPEI.
  5. In the following morning wash the cells with PBS and change the medium.
  6. Let incubate for 48 h after plasmid transfection.
  7. Harvest cells by trypsin treatment.
  8. Separate cells in two tubes. Use half of the cells to prepare cell extracts by adding Laemmli buffer in order to check siRNA effect and I-SceI expression by Western blotting.
  9. Wash the other half of the cells with PBS.
  10. Resuspend in PBS and analyze by flow cytometry to detect GFP positive cells. GFP positive cells represent the cell population in which the DNA double strand break induced at the I-SceI site has been repaired by homologous recombination. Quantify by examining at least 25,000 events per condition.
    Note: First, as a negative control use cells not transfected with the I-SceI plasmid in order to define the window for the detection of negative cells for GFP expression. Then, use a sample of cells transfected with the I-SceI plasmid to obtain a sub population of GFP positive cells that are cells having repaired DNA breaks by homologous recombination (Figure 1).


    Figure 1. Example of FACS acquisition. Left panel, the majority of the cells are GFP negative only 0.06% are GFP positive. Right panel, cells transfected with the I-SceI plasmid, we observe the appearance of a population of GFP positive cells 1.8% present in the R3 region.

Recipes

  1. Laemmli buffer
    Tris HCl pH 6.8 (60 mM)
    10% glycerol
    2% SDS
    5% beta-mercaptoethanol
    0.01% bromophenol blue
  2. Phosphate buffered saline (PBS)
    135 mM NaCl
    2.5 mM KCl
    10 mM Na2HPO4
    1.75 mM KH2PO4

Acknowledgments

This protocol was adapted from previous works by Dumay et al. (2006) and Puget et al. (2005). We acknowledge the use of the Toulouse Rio Imaging facilities for flow cytometry analysis. This work was supported by grants from the Ligue Nationale Contre le Cancer (D. Trouche; équipe labellisée), the Association de Recherche contre le Cancer (ARC) as a Programme ARC, the Agence Nationale pour la Recherche (Projet 2011 blanc SVSE8 PinGs), and by an Electricité de France grant (Y. Canitrot).

References

  1. Courilleau, C., Chailleux, C., Jauneau, A., Grimal, F., Briois, S., Boutet-Robinet, E., Boudsocq, F., Trouche, D. and Canitrot, Y. (2012). The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks. J Cell Biol 199(7): 1067-1081.
  2. Dumay, A., Laulier, C., Bertrand, P., Saintigny, Y., Lebrun, F., Vayssiere, J. L. and Lopez, B. S. (2006). Bax and Bid, two proapoptotic Bcl-2 family members, inhibit homologous recombination, independently of apoptosis regulation. Oncogene 25(22): 3196-3205.
  3. Puget, N., Knowlton, M. and Scully, R. (2005). Molecular analysis of sister chromatid recombination in mammalian cells. DNA Repair (Amst) 4(2): 149-161.

简介

使用由Puget等人开发的含有特异性底物的U2OS细胞或RG37细胞检查通过同源重组修复双链断裂。 (2005)和Dumay等人 (2006),分别通过同源重组来测量DNA双链断裂的修复。 底物由GFP基因的两个无活性拷贝组成。 上游拷贝由于不存在启动子而不活跃,下游拷贝存在启动子,但通过插入编码I-SceI酶的识别位点的序列而失活。 底物在插入基因组后稳定地表达在细胞中,作为唯一拷贝存在。 然后通过用编码I-SceI酶的质粒转染细胞后,通过I-SceI酶的表达来诱导独特的DNA双链断裂。

关键字:DNA双链断裂修复, 同源重组, 人体细胞, DNA修复, 遗传不稳定性

材料和试剂

  1. U2OS或RG37细胞系
    注意:U2OS是具有活性p53途径的骨肉瘤细胞,RG37是具有无活性p53途径的SV40永生化人成纤维细胞。 存在具有设计用于测量同源性定向修复的底物的细胞。 然而,可以使用插入了HDR基板的任何细胞系。
  2. 培养基(无血清培养基)
  3. 用于感兴趣的基因的siRNA(例如 rad51 ,其消耗降低了通过同源重组修复DNA DSB的效率)
  4. 质粒pcDNA3myc-NLS-I-SceI(编码由Puget等人(2005)制造的I-SceI)
  5. INTERFERin转染试剂(Ozyme,目录号:POL409-10)
  6. JetPEI转染试剂(Ozyme,目录号:POL101-10)
  7. 胰蛋白酶
  8. 甘油
  9. SDS
  10. β-巯基乙醇
  11. 溴酚蓝
  12. Laemmli缓冲区(请参阅配方)
  13. 磷酸盐缓冲盐水(PBS)(参见配方)

设备

  1. 35 mm组织培养板
  2. 组织培养(例如37℃,5%CO 2培养箱)
  3. 流式细胞仪
  4. Western印迹仪

程序

  1. 在下午结束时,每35mm直径的平板将100,000个细胞(U2OS或RG37)平板。 在37℃,5%CO 2的细胞培养箱中在潮湿气氛中培养细胞。
  2. 让过夜。
  3. 使用INTERFERIN根据制造商的说明书用siRNA进行转染。 使用10 nM siRNA混合在200μl无血清培养基与8μlINTERFERIN
  4. 24小时后,改变培养基并用编码I-SceI的质粒转染,以在含有I-SceI识别位点的GFP拷贝中诱导DNA双链断裂。 每板使用1μg质粒。 根据制造商的说明书,通过将1μgI-SceI质粒与200μlNaCl(150mM)与2μlJetPEI混合,使用JetPEI进行转染。
  5. 在第二天早上用PBS洗涤细胞并更换培养基
  6. 在质粒转染后孵育48小时
  7. 通过胰蛋白酶处理收获细胞
  8. 在两个管中分离细胞。 使用一半细胞通过加入Laemmli缓冲液以通过Western印迹检查siRNA效应和I-SceI表达来制备细胞提取物。
  9. 用PBS洗涤另一半的细胞。
  10. 重悬于PBS中,通过流式细胞术分析以检测GFP阳性细胞。 GFP阳性细胞代表其中在I-SceI位点诱导的DNA双链断裂已经通过同源重组修复的细胞群体。通过检查每个条件至少25,000个事件进行量化。
    注意:首先,作为阴性对照,使用未用I-SceI质粒转染的细胞,以便定义检测阴性细胞用于GFP表达的窗口。然后,使用用I-SceI质粒转染的细胞样品以获得GFP阳性细胞亚群,其是通过同源重组修复的DNA断裂的细胞(图1)。


    图1.FACS获得的实施例。左图,大多数细胞是GFP阴性,仅0.06%是GFP阳性。右图,用I-SceI质粒转染的细胞,我们观察到在R3区域存在1.8%的GFP阳性细胞群的出现。

食谱

  1. Laemmli缓冲区
    Tris HCl pH 6.8(60mM) 10%甘油 2%SDS
    5%β-巯基乙醇 0.01%溴酚蓝
  2. 磷酸盐缓冲盐水(PBS)
    135 mM NaCl 2.5mM KCl
    10mM Na 2 HPO 4
    1.75mM KH 2 PO 4 sub/

致谢

该协议改编自Dumay等人(2006)和Puget等人(2005)的先前作品。 我们承认使用Toulouse Rio Imaging设备进行流式细胞术分析。 这项工作得到了Ligue Nationale Contrele癌症(D. Trouche;équipelabellisée),癌症研究协会(ARC)作为一个计划ARC,国家民众协会(Projet 2011 blanc SVSE8 PinGs) ,以及法国电力公司(Y. Canitrot)。

参考文献

  1. Courilleau,C.,Chailleux,C.,Jauneau,A.,Grimal,F.,Briois,S.,Boutet-Robinet,E.,Boudsocq,F.,Trouche,D。和Canitrot, 染色质重塑p400 ATP酶促进Rad51介导的DNA双链断裂修复。 J Cell Biol 199(7):1067-1081
  2. Dumay,A.,Laulier,C.,Bertrand,P.,Saintigny,Y.,Lebrun,F.,Vayssiere,J.L.and Lopez,B.S。(2006)。 Bax和Bid,两个促细胞凋亡Bcl-2家族成员,抑制同源重组,独立于凋亡调节。 Oncogene 25(22):3196-3205。
  3. Puget,N.,Knowlton,M。和Scully,R。(2005)。 哺乳动物细胞中姐妹染色单体重组的分子分析 DNA修复) 4(2):149-161。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Canitrot, Y. and Trouche, D. (2013). Homologous Recombination Assay . Bio-protocol 3(18): e914. DOI: 10.21769/BioProtoc.914.
  2. Courilleau, C., Chailleux, C., Jauneau, A., Grimal, F., Briois, S., Boutet-Robinet, E., Boudsocq, F., Trouche, D. and Canitrot, Y. (2012). The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks. J Cell Biol 199(7): 1067-1081.
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