Name: two- and three-dimensional live cell imaging of dna damage response proteins
Uploaded: 2012-09-28T12:00:00
Description: Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

支持部分由R01NS064593和R21ES016636（KV）。显微镜进行VCU - 神经生物学与解剖学显微镜设备，部分支持，资金从NIH-NINDS中心的核心授予5P30NS047463的。旋转盘共聚焦显微镜购买的与NIH-NCRR的奖（1S10RR027957）。

A.细胞制备<ol><li>正常人体主要的成纤维细胞（GM02270）卡瑞尔细胞储存库，卡姆登，新泽西州，并与hTERT 6永生。细胞生长和扩大在蜂星6-cm培养皿在媒体（4毫升）组成的MEM培养基与20％的胎牛血清（GIBCO），的non-essential/essential氨基酸，维生素，丙酮酸钠，和青霉素/链霉素（HyClone公司）。 </li><li>人胚胎肾293（HEK293）细胞，从美国典型培养物保藏中心获得的，并在蜂星6-cm培养皿中生长和?...

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维持基因组的完整性，细胞的生存是至关重要的。如果保存的基因组会导致过早衰老，癌变，死亡8。有浓厚的兴趣，挑剔的DDR功能，源于其基础和临床研究的重要性。多年来，以帮助在细胞如何检测和修复DNA损伤的研究已开发了许多技术。传统的方法，如免疫细胞化学和西方墨点法领域的支柱，尽管最近的技术进步已经使日益复杂的方法发展。活细胞成像，在该协议中详细说明，使我们?...

<table border="1"><tbody><tr><td> 产品 </td><td> 公司 </td></tr><tr><td>陕西蜂星的培养皿</td><td>格雷纳生物一</td></tr><tr><td> FluroDish玻璃底菜</td><td>世界精密仪器公司</td></tr><tr><td> MEM媒体</td><td> GIBCO </td></tr><tr><td>非必需氨基酸</td><td> GIBCO </td></tr><tr><td>氨基酸</td><td> GIBCO </td></tr><tr><td>维生素</td><td> GIBCO </td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td></tr><tr><td>青霉素/链霉素</td><td>胎牛血清</td></tr><tr><td>胎牛血清</td><td> GIBCO </td></tr><tr><td> N-MYC-53BP1 WT pLPC的普罗; 质粒19836 </td><td> Addgene </td></tr><tr><td> pCLNR H2BG质粒17735 </td><td> Addgene </td></tr><tr><td> SuperFect </td><td> Qiagen公司</td></tr><tr><td>蔡司观察SD细胞成像系统</td><td>蔡司</td></tr><tr><td> AxioVision（版本4.8.2） </td><td>蔡司</td></tr><tr><td>蔡司Immersol W油</td><td>蔡司</td></tr><tr><td> Volocity软件（版本6.0） </td><td>珀金埃尔默</td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

双链断裂（DSBs）是最有害的DNA病变的细胞可能会遇到。如果未修复，DNA双链断裂港口的巨大潜力产生突变和染色体畸变1。为了防止这种创伤催化基因组的不稳定性，这是至关重要的，检测DNA双链断裂的细胞，激活DNA损伤反应（DDR），并修复DNA。解除武装，复员和重返社会工作的刺激时，通过触发细胞周期阻滞，以便维修发生或强制的细胞发生凋亡，以维持基因组的完整性。 DSB修复发生的主要机制通过非同源末端连接（NHEJ）和同源重组修复（HRR）（评论2例 ）。有许多的蛋白质，其活动必须精确精心策划的解除武装，复员和重返社会正常运行。这里，我们描述了一种方法，2 - 和3 - 维（D）的可视化这些蛋白质之一，53BP1。 p53结合蛋白1（53BP1）定位于地区DNA双链断裂的修饰组蛋白3,4结合，形成病灶在5-15分钟内5。组蛋白修饰和招聘53BP1和其他DDR蛋白DSB网站被认为是染色质的结构重排，以方便周边地区的损失，并有助于DNA修复6。除了 ​​直接参与修复，额外的角色被描述为53BP1在解除武装，复员和重返社会，如调节内-S检验点，G2 / M检验点，并激活下游DDR蛋白7-9。最近，人们发现，53BP1没有形成病灶在有丝分裂过程中DNA损伤，而不是等待细胞进入G1前定位附近的DNA双链断裂6。如53BP1 DDR蛋白质已被发现与有丝分裂的结构（如着丝粒）相关联的进展通过有丝分裂10期间。 在这个协议中，我们描述了使用2 - 和3-D的活细胞成像的可视化53BP1灶形成的DNA损伤剂喜树碱（CPT），以及53BP1在有丝分裂过程中的行为。喜树碱是拓扑异构酶I抑制剂，主要引起DNA双链断裂DNA复制过程中。要做到这一点，我们使用先前描述的53BP1 mCherry荧光融合蛋白结构的53BP1的蛋白质结构域的能够结合DNA双链断裂11。此外，我们使用组蛋白H2B-GFP的荧光融合蛋白的构建，能够监控整个细胞周期，但在特定的染色质的动态，在有丝分裂12。活细胞成像技术在多个方面，是一个很好的工具，以加深我们对DDR在真核细胞中的蛋白质的功能。

Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

该协议描述了一个可视化的DNA双链断裂以及其定位在有丝分裂过程中DNA损伤激活的信号蛋白的方法。

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Research

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Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

科研

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Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...