Name: 3d multicolor dna fish tool to study nuclear architecture in human primary cells
Uploaded: 2020-01-25T13:00:00
Description: Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

作者感谢INGM成像设施（意大利米兰的"罗密欧·恩弗尼齐"（INGM）的技术援助，特别是C.Cordiglieri在3D多色DNAFISH图像中提供协助。收购。这项工作得到了以下对B.B.的赠款的支持：EPIGEN意大利旗舰计划、法国性心肌病协会（AFM-Telethon，第18754年赠款）和意大利卫生部的吉奥瓦尼·赖斯尔卡托里（GR-2011-02349383）。这项工作得到了以下对F.M.的资助：丰达齐奥内·卡里普洛（班多·乔瓦尼，赠款nr 2018-0321）。

1. DNA探针制备和标签程序，带尼克翻译
<ol><li>净化和清洁细菌人工染色体（BACs），质粒或PCR产品与特定的试剂盒（材料表），重新悬浮在ddH2O，通过电泳检查在胶质和定量。</li>
	<li>根据表1，在50μL的最终体积中混合所有试剂，对步骤1.1的1.5⁄2 μgDNA执行尼克翻译。 注：直接贴标签的探头可以改善信噪比。通过 nick 翻译，用于生产带有各种 Alexa 氟铬的间接和?...

<ol>
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目前的方法描述了一个分步协议，以在广泛的人类原细胞上执行3D多色DNAFISH。虽然DNA FISH是一项广泛使用的技术，但保存下来的3D相间核上的3D多色DNAFISH在许多实验室中仍然难以执行，这主要是因为使用23、24的样品的特性。
探针刻翻译是成功3D多色DNAFISH的基本步骤;许多不同的基板（BAC、fosmid、质粒、PCR产物）可用于这种反应，反应...

较高的真核细胞需要系统地压缩和压缩大量的遗传信息在一分钟的3D空间的细胞核1，2，3，4。今天，我们知道基因组在空间上按隔间和拓扑相关域5排序，DNA折叠的多级在不同基因组区域之间产生接触，可能涉及染色质环形成6，7。染色质的3D动态循环可以影响许多不同的生物过程，如转录8、9、分化和发育10、11、DNA修复12、13，而其扰动涉及各种疾病14、15、16和发育缺陷15、17、18。
已经开发出许多方法来研究3D基因组组织。染色体构象捕获技术（C技术，3C，4C，5C，高C和衍生物）已经开发，以研究基因组组织在固定细胞3，4，19，20。这些方法基于在物理接近的基因组位点之间捕获接触频率的能力。C-技术，根据其复杂性，捕捉全球3D基因组组织和细胞群的3，4，19，20的核拓扑。然而，3D交互在时间和空间上是动态的，由多路复用相互作用组成的单个细胞之间的高度可变，并且广泛是异质的21，22。
3D 多色DNA 荧光原位杂交 （FISH） 是一种允许在单个细胞级别上可视化特定基因组位点的技术，能够以与 C 技术互补的方式直接研究 3D 核架构。它表示当前用于明确验证 C 结果的技术。3D 多色 DNA FISH 使用荧光标记探针，与兴趣的基因组位点互补。使用不同的荧光镜和合适的显微镜设备，使核空间23、24内多个目标的当代可视化。近年来，FISH与显微镜技术的进步相结合，获得了高分辨率25、26或CRISPR-Cas方法的精细结构可视化，用于在活成像27、28中实现核酸可视化。尽管广泛采用，3D 多色DNA FISH 方法在许多实验室中仍然被认为是困难的，因为所使用的生物材料必须加以调整。
在这里，我们为适用于各种人类原细胞的3D多色DNAFISH（从细胞/探针制备到数据分析）提供全面协议，实现多个基因组位点的可视化并保留核的3D结构。为了研究核结构，必须保留核的三维结构。因此，与其他现有协议29，30，31相比，我们避免使用酒精梯度和酒精中可能影响染色质结构32的盖玻片。该方法根据保存的3DDNAFISH协议24，33改编，适用于广泛的人类原细胞，包括分离的体外或体外培养。不同的核形态和细胞学特征（例如，不同程度的核压实、细胞骨架丰度）有渗透和脱蛋白参数34。这些参数通常在其他协议24，33中描述，但没有提供不同细胞类型中程序的明确区分。此外，我们开发了一个名为NuCL_D（3D中的核接触定位器）16的特定工具，为数据分析提供了原理，从而以自动方式改进不同位点之间的3D接近及其核拓扑分布。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin 488</td>
			<td>DBA</td>
			<td>DI7488</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin 11 d-UTP</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0081</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverlsips</td>
			<td>Marienfeld</td>
			<td>117500</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 d-UTP</td>
			<td>GE Healthcare</td>
			<td>PA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D21490</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acids single strand from salmon testes</td>
			<td>Sigma</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate (powder)</td>
			<td>Santa Cruz</td>
			<td>sc-203917A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin 11 d-UTP</td>
			<td>Roche</td>
			<td>11093088910</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21969-035 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>18010-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>AMPD1</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (C-G-A-T)</td>
			<td>Euroclone</td>
			<td>BL0423A/C/G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644.2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Hyclone</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30109</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutammine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Human</td>
			<td>Sigma</td>
			<td>I9278-5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate-buffered saline)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennycillin/Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Biorad</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>PhasePrep BAC DNA Kit</td>
			<td>Sigma</td>
			<td>NA0100-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Gel Extraction &#38; PCR Purification Combo Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K220001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2288</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbercement</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>631-0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidina Alexa fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>S21374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate</td>
			<td>Sigma</td>
			<td>1110379026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P9416-100mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

细胞生物学的一个主要问题是核空间内的基因组组织，以及染色质结构如何影响基因表达、细胞分化和分化等过程。研究基因组的3D架构的许多方法可以分为两个互补的类别：基于染色体构象捕获的技术（C技术）和成像。前者基于捕获固定细胞群中的染色体构象和近端DNA相互作用，而后者基于DNA荧光原位杂交（FISH）的3D保存核，允许当代可视化单个细胞水平（多色）中的多个位点，检查其在细胞核内的相互作用和分布（3D多色DNA FISH）。3D多色DNAFISH技术只对几个预定位点进行可视化，无法对核结构进行全面分析。然而，鉴于其结果的鲁棒性，3D多色DNA FISH与3D显微镜和图像重建相结合是验证基于C技术的结果和明确研究特定位点的位置和组织的可能方法在单个单元格级别。在这里，我们提出了一个适合广泛的人类原细胞的3D多色DNAFISH的一步一步方法，并讨论了获得成功和翔实的3D多色所需的所有实际行动、关键步骤、3D成像概念和数据分析不同生物背景下的DNAFISH。

本文中描述的三维多色DNA FISH方法允许当代可视化保存的3D核内的不同基因组位点（图1B）。该协议允许测量等位基因和不同基因组位点之间的距离，以评估其空间接近度，并评估其在核空间内的位置（例如，离心源或核外围的位点距离）16。但是，有许多关键步骤必须针对所使用的每种单元格类型进行准确和专门设置;强烈建议特别注意以下步?...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D 多色 DNA FISH 是一种工具，用于可视化 3D 保留核内的多个基因组位点，明确定义其在单个细胞级别的核空间内的相互交互和定位。在这里，描述了一个分步协议，用于广泛的人类原细胞。

3D多色DNAFISH工具研究人类原细胞核结构

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

3D 多色 DNA FISH 能够可视化保存的核内的多个基因组位点，从而模糊地定义它们在单细胞级别的相互相互作用和定位。3D多色DNA鱼允许直接研究核结构。通常，它与基于染色体捕获的技术结合使用，使该技术成为单细胞内C数据验证的可用工具。演示程序将是费德里卡马拉斯卡。她是我实验室的博士后首先根据起始DNA材料的长度在16摄氏度的热混合器中孵育刻痕转换混合物。在孵育结束时，检查2.2%的加糖凝胶上的探头尺寸。对于每个DNA鱼实验， 根据起始DNA材料，在150微升双蒸馏水中生产探针，20微克未标记鲑鱼精子DNA，3.5微克特定Cot-1 DNA，三卷100%乙醇，以及1/10体积的3个醋酸钠，在零下80摄氏度下1小时。在孵育结束时，以最高速度将样品在4摄氏度下离心一小时。丢弃上经剂后，用500微升70%乙醇洗涤颗粒两次。第二次洗涤后，将颗粒重新注入两个微升100%甲酰胺中，并在40摄氏度下摇动探针30分钟，然后加入20%脱酸硫酸盐中等量的4X盐水柠檬酸钠。对于小核小细胞和少量细胞质的小细胞的固定预处理和渗透，首先在PBS200微升中的第六细胞中加入2倍10，直接添加到24孔板的单个孔的玻璃盖玻片上，使细胞在室温下沉淀30分钟。在孵育结束时，快速更换PBS与300微升新鲜准备的4%半成甲醛，并辅以0.1%Tween 20。10分钟后，在300微升0.05%TPBS中三次清洗固定细胞，在室温下每次清洗5分钟。上次洗涤后，在室温下用 300 微升 0.5 TPBS 渗透 T 细胞 10 分钟，然后用每井 250 微升的 RNAse 鸡尾酒在 PBS 中稀释 1：100，在 37 摄氏度下稀释 1：100。在孵育结束时，在300微升PBS中冲洗细胞，并在每口井中加入300微升20%甘油，并在4摄氏度下进行隔夜孵育。第二天早上，将玻璃放在干冰上15至30秒，冷冻细胞，然后逐渐在室温下解冻样品，并在PBS中浸泡在300微升20%甘油中30秒。冷冻/解冻细胞三次后，在室温下用300微升0.5%TPBS清洗样品5分钟，随后在300微升中清洗两次0.05%TPBS，在室温下每次清洗5分钟。最后一次洗涤后，用300微升0.1法兰酸在室温下处理细胞12分钟，然后用300微升盐水柠檬酸微升进行两次冲洗。然后在室温下将样品在300微升中，在2X盐水柠檬酸钠中固定50%的酰胺。对于具有大量细胞质的大细胞的固定、预处理和渗透，固定、预处理和渗透细胞与人体原生T细胞相似，并在0.01普通盐酸中用300微升0.0025%辣椒处理样品，几秒钟至5分钟。在光学显微镜下观察细胞，并在核从细胞质中释放，但核仍然可见且完好无损，在300微升50毫摩尔氯化镁中停止反应。对于 3D 多色 DNA FISH，将杂交探测器在 80 摄氏度下变性 5 分钟，并立即将探测器放在冰上。将探头加载到干净的显微镜幻灯片上，并在每滴探针上放置一个细胞盖玻片。用橡胶水泥密封盖玻片边缘。当水泥完全干燥后，将样品在 75 摄氏度的加热块上变性。4分钟后，在37摄氏度的温度下，在漂浮在水浴中的金属盒中对样品进行水化。第二天早上，剥下橡胶水泥，用滑梯浸入2倍盐水柠檬酸钠中，小心地从盖玻片上抬起来。将每个盖玻片放入六孔板的单个孔中，每口井含有两毫升新鲜盐水柠檬酸钠，在 37 摄氏度下进行两次 5 分钟的洗涤，每分钟摇晃 90 圈。在孵育结束时，在4X盐水柠檬酸钠中用0.2%Tween 20的两毫升短暂冲洗盖玻片。对于 3D 多色 DNA FISH 检测，将盖玻片转移到新的 24 孔板的单个孔中，并在 300 微升阻塞缓冲液中阻断任何非特异性结合，在 37 摄氏度和每分钟 20 转下进行 20 分钟。在孵化结束时，在37摄氏度的黑暗湿润室中，用适当浓度的抗二氮二氮素和/或链球菌素在阻尼缓冲液中稀释35分钟。在孵育结束时，将样品转移到六井板的单个孔中，用两毫升0.2%Tween 20和4X盐水柠檬酸钠洗涤三次，每次洗涤5分钟，每分钟摇晃90圈。上次洗涤后，将样品平衡在两毫升 PBS 中，然后将盖玻片转移到新的 24 井板中，在 300 微升中固定 300 微升，每井 PBS 中甲醛为 2%，在室温下两分钟。在 300 微升 PBS 中短暂清洗固定细胞五次，随后在室温下用 DAPI 稀释，每毫升一毫微克，在 300 微升 PBS 中洗涤 5 分钟。在 PBS 中多次清洗盖玻片，然后使用适当的安装介质安装样品。然后通过显微镜系统获取 3D 图像。3D 多色 DNA FISH 允许在保存的 3D 核中对不同基因组位进行当代可视化。对于DNA探针制备，小于200个碱基对的探针大小确保了成功的3D多色DNA FISH程序。由刻痕翻译产生的不理想的DNA FISH探针可以部分或过度消化。过度消化的探头会导致非特异性信号，因为杂交中特异性的丧失以及背景的随从增加。对于具有小核和低细胞质的人类原发性T淋巴细胞和小细胞，建议进行12分钟0.1法线盐酸处理，以促进核对DNA探针的可访问性，并维护核完整性。细胞质消化不需要获得DNA鱼良好的信号在小细胞。然而，对于具有大核和丰富细胞质的人类原发性肌细胞和细胞来说，百事可乐化步骤是根本的。短和次优的细胞骨架百化将阻碍探针进入核结束，没有DNA FISH信号。然而，如果细胞过度化，核不会保持完好无损，失去他们的3D结构。一个成功的3D多色DNA鱼将导致在核内的信号存在。我建议使用新鲜的生物材料，在执行三维多色DNA鱼之前测试探针，准备新的解决方案，并精确测量孵化时间和温度。请记住，甲酰胺和 HCL 是有害物质，应始终用于化学烟罩中，并配有适当的一次性材料。

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

原发性人类 CD4+ T 淋巴细胞低线粒体 DNA 污染的 ATAC-seq 检测

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

多点PCR和毛细血管电泳对鱼类致病菌的多点变量数串联重复分析

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

导航 MARRVEL,一种基于 Web 的工具,集成了人类基因组学和模型有机体遗传学信息

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...