Name: monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy
Uploaded: 2012-12-13T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

这项工作是由美国国立卫生研究院和ACS，包括T32CA009135，CA133027，CA070723，和CA022443补助。比尔萨格登是美国癌症学会的研究教授。

1。可见质粒的细胞工程<ol><li>使用标准的限制性消化或同源重组技术引入到质粒加以可视化的DNA片段，它含有约250份的乳糖操纵子序列（LACO）。我们的质粒Bacmid的是一个约170 kbp的，并包含了整个基因组的卡波济氏肉瘤相关疱疹病毒（KSHV）编码的抗潮霉素12。 </li><li>介绍法律咨询及田土转易处含有质粒DNA，脂质体2000转染哺乳动物细胞中。转染HeLa细胞和SLK细胞在60毫米菜为4小时10μg纯化?...

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这里描述的方法可以被用来追踪跨越多个世代的随着时间的推移在活的哺乳动物细胞中的质粒。通过限制暴露于激发光，我们已经使用了这些技术，按照新划分对16-32细胞的殖民地，在至少72个小时和3个细胞分裂的细胞。这些实验提供不能获得的信息，可以从静态试验，如定量PCR，Southern杂交，和FISH。 关键是要筛选的细胞克隆具有均匀的结构的质粒，作为重排时，可能会发生...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11965 </td><td></td></tr><tr><td> OptiMEM </td><td> GIBCO </td><td> 31985 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30910.03 </td><td></td></tr><tr><td>青霉素</td><td>西格玛</td><td> P3032 </td><td></td></tr><tr><td>硫酸链霉素</td><td>西格玛</td><td> S9137 </td><td></td></tr><tr><td>潮霉素</td><td> Calbiochem公司</td><td> 400050 </td><td> 400微克/毫升的SLK; 300微克/毫升的HeLa细胞</td></tr><tr><td>异丙基-BD-硫代半乳糖苷（IPTG） </td><td>罗氏公司</td><td> 10 724 815 001 </td><td>最终浓度200微克/毫升</td></tr><tR&gt; <td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668-027 </td><td></td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td></td></tr><tr><td>玻璃底菜</td><td> MatTek </td><td> P35G-1.5-10-C </td><td></td></tr><tr><td> CultFoil </td><td> Pecon </td><td> 000000-1116-08 </td><td></td></tr><tr><td> Axiovert 200M </td><td>蔡司</td><td></td><td></td></tr><tr><td>科利柏</td><td>蔡司</td><td> 423052-9500-000 </td><td></td></tr><tr><td>中性白色LED </td><td>蔡司</td><td> 423052-9120-000 </td><td></td></tr><tr><td>过滤器立方75 HE </td><td>蔡司</td><td> 489075-0000-000 </td><td></td></tr><tr><td>计划复消色差63x/1.4目标</td><td>蔡司</td><td> 440762-9904-000 </td><td></td></tr><tr><td> CascadeII：1024 EMCCD相机</td><td>光度</td><td> B10C892007 </td><td></td></tr><tr><td> Tempcontrol小</td><td>蔡司/ Pecon </td><td> 000000-1116-070 </td><td></td></tr><tr><td> Tempcontrol 37-2数字</td><td>蔡司/ Pecon </td><td> 000000-1052-320 </td><td></td></tr><tr><td> CTI控制器3700数字</td><td>蔡司/ Pecon </td><td> 411856-9903 </td><td></td></tr><tr><td>目的加热器</td><td>蔡司/ Pecon </td><td> 440760-0000-000 </td><td></td></tr><tr><td>第一阶段加热插入P </td><td>蔡司/ Pecon </td><td> 411861-9901-000 </td><td></td></tr><tr><td>舞台顶部孵化器小号</td><td>蔡司/ Pecon </td><td> 411860-9902-000 </td><td></td></tr><tr><td>加湿系统</td><td>蔡司/ Pecon </td><td> 000000-1116-065 </td><td></td></tr></tbody></table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

保持在哺乳动物细胞中很少有天然存在的质粒。其中γ-疱疹病毒的基因组，包括Epstein-Barr病毒（EBV）和卡波济氏肉瘤相关疱疹病毒（KSHV），这会导致多种人类恶性肿瘤的1-3。在有执照的方式，每使用一个单一的病毒蛋白和细胞复制机制，这两个基因组复制传递到子代细胞在细胞分裂过程中，尽管他们缺乏传统的着丝粒4-8。 已经做了许多工作，这些质粒基因组的复制特性，使用方法，如Southern杂交和荧光原位杂交（FISH）。是有限的，虽然这些方法。定量PCR和Southern杂交提供的每个细胞质粒细胞的人口平均数的信息。 FISH技术是一种单细胞的检测，揭示的平均数量和分布的质粒<html> s的细胞的细胞群，但是是静态的，允许任何信息的父母或研究细胞的后代。 在这里，我们描述了一个可视化的质粒在活细胞中的方法。这种方法是基于上的荧光标记的景点9的质粒中的多个站点的乳糖阻遏蛋白的结合。感兴趣的DNA的设计，以约250包括乳糖运算符（LACO）序列的串联重复序列。 LACO特异性结合由乳糖阻遏蛋白（LacI的），它可以是稠合的荧光蛋白质。的融合蛋白可以从工程质粒或引进由逆转录病毒载体表达。在这种方式中，该DNA分子是荧光标记的，并因此成为可见的，通过荧光显微镜。的融合蛋白被阻止质粒是通过在IPTG存在下培养细胞中的质粒DNA的绑定，直到准备好以进行查看。 ontent“&gt;此系统允许被监视的质​​粒通过几代人在活细胞中，揭示其合成的属性和分区子细胞。理想的细胞粘附，容易转染，并具有大的细胞核。这种技术已经被使用，以确定84％的EBV-来源质粒合成每一代和88％的新合成的质粒分区忠实地对这些EBV质粒在HeLa细胞中的子细胞。看到被拴或与姐妹染色单体在S之后所合成阶段，直到他们被视为分开的姐妹染色单体分离，后期10的方法目前正在研究的KSHV在HeLa细胞中的基因组和SLK细胞的复制。HeLa细胞的永生化人上皮细胞，SLK细胞永生化人脐静脉内皮虽然SLK细胞最初来自一个KSHV病变的细胞。，本HeLa细胞也SLK细胞系自然哈布口服补液盐KSHV基因组的11。除了研究病毒的复制，该可视化技术可用于调查其他重组质粒DNA的合成，定位，和分区的各种DNA序列上的元素的添加，删除，或突变的影响。

<html>在图3中示出在一个有代表性的实验中获得的z-堆栈的最大强度投影。典型的质粒的信号都出现在3-8片的z-堆栈，这取决于它们是否代表单或簇的质粒。在EBV-和KSHV衍生质粒的情况下，在细胞内信号慢慢地移动，直到电池达到的有丝分裂。细胞本身迁移的实验过程。他们也变成球形，并在有丝分裂过程中脱离的菜。健康的HeLa细胞和SLK细胞的细胞周期约24小时，;这些类型超过30小时，?...

Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

观察单个活细胞中的DNA分子的方法，进行说明。该技术是基于对一个荧光标记的lac阻遏物蛋白质设计到感兴趣的DNA结合位点的结合。此方法可适于后续许多随着时间的推移在活细胞中的重组DNA。

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Research

JoVE Journal

Biology

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...