Name: assessment of global dna double-strand end resection using brdu-dna labeling coupled with cell cycle discrimination imaging
Uploaded: 2021-04-28T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging at JoVE.com

我们感谢Marie-Christine Caron出色的技术建议。这项工作得到了加拿大卫生研究院J.Y.M（CIHR FDN-388879）的资助。J.-Y.M.在DNA修复和癌症治疗方面拥有加拿大一级研究主席。J.O'S是FRQS博士生研究员，S.Y.M是FRQS博士后研究员。

1. 细胞培养、处理和盖玻片制备
注意：除照射外，所有细胞接种、转染和治疗均应在无菌细胞培养罩下进行。
<ol><li>第 1 天<ol>
<li>在6孔板中，在每个孔中放置一个盖玻片，以根据需要满足多种条件。根据需要将约 150，000 个 HeLa 细胞用于转染或药物治疗。 注意：如果转染，建议在接种时进行反向转染，或者可以在接种后数小时进行正向转染以允许粘附。反向转染是通过?...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+killing+of+BRCA2-deficient+tumours+with+inhibitors+of+poly(ADP-ribose)+polymerase.">Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.</a> Nature. 434 (7035), 913-917 (2005).</li><li>Farmer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+DNA+repair+defect+in+BRCA+mutant+cells+as+a+therapeutic+strategy.">Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.</a> Nature. 434 (7035), 917-921 (2005).</li><li>Zhou, P., Wang, J., Mishail, D., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+PARP+inhibitors-based+targeted+cancer+therapy.">Recent advancements in PARP inhibitors-based targeted cancer therapy.</a> Precision Clinical Medicine. 3 (3), 187-201 (2020).</li><li>Noordermeer, S. M., van Attikum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+inhibitor+resistance:+a+tug-of-war+in+BRCA-mutated+cells.">PARP inhibitor resistance: a tug-of-war in BRCA-mutated cells.</a> Trends in Cell Biology. 29 (10), 820-834 (2019).</li><li>Caron, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribose)+polymerase-1+antagonizes+DNA+resection+at+double-strand+breaks.">Poly(ADP-ribose) polymerase-1 antagonizes DNA resection at double-strand breaks.</a> Nature Communications. 10 (1), 2954 (2019).</li><li>Zhou, Y., Caron, P., Legube, G., Paull, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+double-strand+break+resection+intermediates+in+human+cells.">Quantitation of DNA double-strand break resection intermediates in human cells.</a> Nucleic Acids Research. 42 (3), 19 (2014).</li><li>Raderschall, E., Golub, E. 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本文描述了一种利用IF染色来测量 纤维素中DNA切除变异的方法。目前观察对DNA切除的影响的标准是通过RPA染色;然而，这是一种可能受DNA复制影响的间接方法。此前，已经描述了另一种基于BrdU掺入的DNA切除IF技术，用于对BrdU阳性和BrdU阴性细胞中产生的强度进行分类。该方法允许由于背景或线粒体染色而未进行HR的细胞被计为阳性，从而导致高BrdU强度14，<sup class="xre...

DNA修复因子的调节是癌症治疗的一种不断发展的方法，特别是在DNA DSB修复缺陷的肿瘤环境中。抑制特定修复因子是用于使癌细胞对DNA损伤剂敏感的巧妙策略之一。数十年的研究导致鉴定出DNA修复基因的各种突变作为治疗策略选择的生物标志物1。因此，DNA修复领域已成为药物开发的中心，以确保广泛的治疗，从而增强个性化医疗概念。 
DSB通过两种主要途径进行修复：NHEJ和HR2。NHEJ途径容易出错，快速连接两个DNA末端，几乎没有DNA末端处理，涉及蛋白激酶（DNA-PKcs），Ku70 / 80复合物，53BP1和RIF1蛋白3。相比之下，HR是由BRCA14发起的忠实机制。HR修复的一个重要步骤是DNA末端切除过程，该过程是断裂末端的降解，导致具有3'-OH末端的单链（ss）DNA。BRCA1 促进下游蛋白的募集，这些蛋白形成分离体 MRN/RPA/BLM/DNA2/EXO1，这些蛋白参与 5' 至 3' DNA 切除5。 
初始末端切除是通过MRE11的内切酶活性完成的，允许DNA2和EXO1核酸酶进一步处理。生成的ssDNA悬垂被复制蛋白A（RPA）快速包被，以保护它们免受进一步处理。随后，BRCA2，PALB2和BRCA1接合以介导RPA的位移和同源定向修复机制所需的RAD51核纤维的组装。NHEJ和HR的使用之间的良好平衡对于基因组完整性的最佳维护是必要的。通路的选择取决于细胞周期阶段。HR优先用于S至G2阶段，其中DNA切除处于最高水平，并且姐妹染色单体可用于确保适当的修复。
聚（ADP-核糖）聚合酶1（PARP-1）是最早招募到DSB的蛋白质之一。它调节切除活性和NHEJ5，6中涉及的下游效应器的组装。在复制过程中，DNA单链断裂修复也需要PARP-17，8。由于其在DNA修复中的重要作用，PARP抑制剂（PARPi）被用作癌症疗法。在几种HR缺陷癌症中，PARPi治疗导致合成致死反应，因为HR缺陷细胞无法通过替代途径修复累积的损伤9，10。目前有四种FDA批准的PARPi：Olaparib，Rucaparib，Niriparib和Talazoparib（也称为BMN 673），用于各种乳腺癌和卵巢癌治疗11。然而，PARPi 耐药性很常见，一个潜在的原因就是通过重新获得 HR 熟练度12 而产生的。在存在辐照的情况下，PARP-1的丢失或抑制会使切除体机制失调，导致更长的ssDNA束积累13。因此，深入研究体内DNA切除对于更清楚地了解DNA修复途径以及随后开发治疗癌症和克服PARPi耐药性的新策略至关重要。
已经有几种方法用于检测DNA切除事件5。其中一种方法是基于IF的经典技术，允许使用抗RPA抗体在应激诱导的DSB后对切除的DNA进行间接染色和可视化。用5-溴-2′-脱氧尿苷（BrdU）标记基因组DNA并仅检测ssDNA是对DNA切除事件的直接测量。它规避了RPA的监测，RPA涉及DNA复制等多个细胞过程。在这里描述的方法中，与BrdU一起孵育的细胞进行单个细胞周期允许BrdU掺入复制细胞DNA的一条链中。切除后，在允许仅以ssDNA形式检测BrdU的条件下进行IF染色，并使用抗BrdU抗体。这种抗体只能进入暴露的BrdU核苷酸，不会检测到那些整合到双链DNA中的核苷酸。使用荧光显微镜，切除的DNA可以以点状BrdU / ssDNA病灶的形式可视化。这些病灶的核强度可用作量化DNA损伤后切除的读数。本文逐步描述了该方法的过程，该方法可以应用于大多数哺乳动物细胞系。这种方法应该具有广泛的实用性，作为监测 纤维素中DNA末端切除的简单方法，作为概念证明。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa 568 goat anti-rabbit</td>
			<td>Molecular probes</td>
			<td>A11011</td>
			<td>1:800</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse</td>
			<td>Molecular probes</td>
			<td>A11001</td>
			<td>1:800</td>
		</tr>
		<tr>
			<td>Anti PARP1 (F1-23)</td>
			<td>Homemade</td>
			<td></td>
			<td>1:2500</td>
		</tr>
		<tr>
			<td>Anti PCNA (SY12-07)</td>
			<td>Novus</td>
			<td>NBP2-67390</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-Alpha tubulin (DM1A)</td>
			<td>Abcam</td>
			<td>Ab7291</td>
			<td>1:100000</td>
		</tr>
		<tr>
			<td>anti-BrdU</td>
			<td>GE Healthcare</td>
			<td>RPN202</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Benchtop X-ray Irradiator</td>
			<td>Cell Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMN673</td>
			<td>MedChem Express</td>
			<td>HY-16106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU)</td>
			<td>Sigma</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell profiler</td>
			<td>Broad Institute</td>
			<td>V 3.19</td>
			<td>https://cellprofiler.org/</td>
		</tr>
		<tr>
			<td>Curwood Parafilm M Laboratory Wrapping Film 4in / 250 ft</td>
			<td>Fisher scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen Life Technology</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Fisher scientific</td>
			<td>10063542</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Squares 22 x 22</td>
			<td>Fisher scientific</td>
			<td>12 541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DMI6000B</td>
			<td>63x immersion objective</td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL 160I CO2 INCUBATOR CU 1-21 TC 120V</td>
			<td>VWR</td>
			<td>51030408</td>
			<td>37% CO2</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>BioShop Canada</td>
			<td>MAG520.500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioShop Canada</td>
			<td>SOD002.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Wisent Bio Products</td>
			<td>311-010-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Cedarlane Labs</td>
			<td>15710-S(EM)</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen Life Technology</td>
			<td>P-36930</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAiMAX</td>
			<td>Invitrogen</td>
			<td>13778-075</td>
			<td></td>
		</tr>
		<tr>
			<td>siPARPi</td>
			<td>Dharmacon</td>
			<td></td>
			<td>AAG AUA GAG CGU GAA GGC GAA dTdT</td>
		</tr>
		<tr>
			<td>siRNA control</td>
			<td>Dharmacon</td>
			<td></td>
			<td>UUCGAACGUGUCACGUCAA</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholcate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop Canada</td>
			<td>SUC507.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>BioShop Canada</td>
			<td>TRS001.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trition X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher scientific</td>
			<td>BP337500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of global dna double-strand end resection using brdu-dna labeling coupled with cell cycle discrimination imaging

DNA损伤反应（DDR）的研究是一个复杂而重要的领域，由于使用DDR靶向药物进行癌症治疗，这一领域变得更加重要。这些靶标是聚（ADP-核糖）聚合酶（PARPs），其启动各种形式的DNA修复。使用PARP抑制剂（PARPi）抑制这些酶，通过在乳腺癌1型（BRCA1），BRCA2或BRCA2（PALB2）的伴侣和定位剂的突变中赋予同源重组（HR）缺陷细胞的治疗脆弱性来实现合成致死性。 
用PARPi处理的细胞积累DNA双链断裂（DSBs）。这些断裂由DNA末端切除机制处理，导致单链（ss）DNA的形成和随后的DNA修复。在BRCA1缺乏的情况下，通过DNA切除抑制剂（如53BP1和DYNLL1）的突变重新激活DNA切除，会导致PARPi耐药性。因此，能够监测 纤维素中的 DNA切除对于更清楚地了解DNA修复途径和开发克服PARPi耐药性的新策略至关重要。基于免疫荧光（IF）的技术允许在DNA损伤后监测整体DNA切除。该策略需要使用5-溴-2′-脱氧尿苷（BrdU）进行长脉冲基因组DNA标记。在DNA损伤和DNA末端切除术之后，产生的单链DNA在天然条件下被抗BrdU抗体特异性检测。此外，还可以使用细胞周期标记物研究DNA切除，以区分细胞周期的各个阶段。S / G2期的细胞允许研究HR内的末端切除术，而G1细胞可用于研究非同源末端连接（NHEJ）。本文描述了这种IF方法耦合到细胞周期判别的详细方案。

在该协议中，基于溴脱氧尿苷（BrdU）的测定用于定量测量HeLa细胞对照射诱导的损伤的切除反应。生成的ssDNA轨迹在免疫荧光染色后被可视化为不同的病灶（图1A）。然后将确定的病灶量化并表示为细胞核中BrdU染色的总综合强度（图1B，补充图S1，补充图S2 和 补充图S3）。可以测量病灶数或平均病灶强度，尽管这可能不如总核强?...

Watch this Scientific Journal Video about Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging at JoVE.com

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

在本方案中，我们展示了如何使用基于免疫荧光的方法在细胞周期的S / G2阶段可视化DNA双链末端切除。

使用BrdU-DNA标记结合细胞周期鉴别成像评估全球DNA双链末端切除术

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

该协议允许在纤维素中对DNA进行定量，并在DNA损伤后进行切除。该技术的主要优点是细胞周期标记，将细胞限制在S和G2相，确保溴脱氧尿苷信号来自DNA和切除。演示该程序的将是首席研究员Jean-Yves Masson和博士后研究员Sofiane Y.Mersaoui。在无菌培养罩下工作，在六孔板的每个孔中放置一个盖玻片，以达到所需的条件。平板约150， 000个HeLa细胞用于转染或药物治疗。在第三天，在用BrdU以10微摩尔的终浓度孵育过夜后，用总剂量为5灰度的X射线照射照射板。照射后，将板返回在37摄氏度下在5%氧化碳加湿的培养箱中孵育三小时。对于预提取，吸出培养基并用PBS仔细洗涤细胞两次。除去PBS后，加入两毫升预提取缓冲液A，并将细胞在冰上以4摄氏度孵育10分钟。10分钟后，吸出缓冲液A，加入2毫升细胞骨架剥离缓冲液B，并重复孵育。然后小心地吸出缓冲液B并用PBS洗涤细胞。吸入PBS后，通过在化学罩下添加两毫升4%的多聚甲醛来固定细胞。除去多聚甲醛并用PBS洗涤后，用100%冷甲醇盖住盖玻片，在零下20摄氏度下孵育五分钟，并用PBS洗涤盖玻片两次。为了透化，在室温下用两毫升含有0.5%Tritan X-100的PBS孵育细胞。15分钟后，用两毫升PBS清洗盖玻片三次。对于免疫染色，向每个孔中加入两毫升封闭缓冲液。孵育一小时后，向每个盖玻片中加入100微升一抗溶液。使用镊子用方形的石蜡片覆盖盖玻片，并小心地放置胶片，以免产生气泡。然后将板覆盖在铝箔中，并将一抗在加湿室中以4摄氏度孵育过夜。第二天，取出石膏板方块，用两毫升PBS清洗盖玻片三次。洗涤后，在每个盖玻片上加入100微升二抗溶液，并用一块正方形的副膜覆盖盖玻片，如图所示。将二抗在室温下孵育一小时，并使用箔保护板免受光照。然后用两毫升1X PBS洗涤盖玻片三次。对于核染色，在每个盖玻片中加入两毫升DAPI溶液，并用PBS清洗盖玻片。在显微镜载玻片上加入10至20微升IF特异性安装介质。使用针或细镊子从井底抬起盖玻片。通过轻轻拍打纸巾上的一个边缘，小心地吸走多余的液体，并将盖玻片安装在显微镜载玻片上。对于图像采集和分析，请将这些文件导入CellProfiler并使用斑点计数管道对其进行分析。使用文本手稿中描述的设置确定主要对象以识别焦点并测量强度。这里显示了照射后5-溴-2-prime-脱氧尿苷病灶形成和增殖细胞核抗原染色的代表性图像。生成的单链DNA轨迹被可视化为不同的病灶。然后将鉴定的病灶量化并表达为细胞核中溴脱氧尿苷染色的总综合强度。共染色显示，由于非同源末端连接而导致的短程切除与使用抗PCNA抗体鉴定通过S期的细胞的同源重组的长距离切除之间存在差异。PCNA在细胞核中突出，在细胞周期的S期达到最大表达，染色相当强烈。然后将值中的结果绘制为散点图，以区分PCNA阳性和PCNA阴性核。PCNA阴性结果从数据集中删除，以便进行基于BrdU强度的分析，因为无论条件如何，BrdU信号都很低。PCNA阴性核具有900个任意单位的BrdU病灶的基础综合强度，该值在PCNA阳性核中达到1， 800 AU。在siRNA控制条件下，PCNA阳性细胞核中每个细胞核的BrdU病灶的综合强度约为1， 500 AU。在使用siRNA有效敲低PARP-1后，观察到BrdU病灶的强度显着增加至约1， 900 AU。该技术可以为同源重组的第一阶段提供关键见解，从而有助于确定蛋白质是否参与修复途径的第一步。必须严格保持预提取缓冲液的孵育时间。过度暴露于这些缓冲液会导致过度的细胞脱离和增加的背景信号。

Research

JoVE Journal

Biology

Measuring Exocytosis in Neurons Using FM Labeling

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

使用定量成像技术鉴定和表征异源细胞群体

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...