Name: measuring the kinetics of mrna transcription in single living cells
Uploaded: 2011-08-25T13:00:00
Description: Watch this Scientific Journal Video about Measuring the Kinetics of mRNA Transcription in Single Living Cells at JoVE.com

YS - T是由欧洲研究委员会（ERC），以色列科学基金会（ISF）（250/06），ISF Bikura，以色列癌症研究基金会（ICRF），德国和以色列的科技研究和开发基金会（GIF的支持），美国和以色列两个民族组成的科学基金会（BSF），德国和以色列项目合作（DIP），和以色列科学和健康的部委，并在生命科学，简斯特恩Lebell家庭巴伊兰大学的研究员。 YB是Azrieli Azrieli奖学金奖基金会表示感谢。

所使用的电池系统必须包含一个集成的基因构造，包括MS2的重复序列标记在活细胞中的mRNA。在这个协议中，我们使用人类U2OS细胞株窝藏一个稳定的集成β-肌动蛋白基因，荧光标记的DNA，RNA和蛋白水平8，如下（图1A）：该基因的5&#39;包含lac操纵子（ 法律咨询及田土转易处）重复-这是用来标记基因（DNA），从而确定基因组位点的整合。一个荧光标记的lac阻遏蛋白（LACI）...

<ol>
	<li>Wada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wave+of+nascent+transcription+on+activated+human+genes.">A wave of nascent transcription on activated human genes.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 18357-18361 (2009).</li><li>Singh, J., Padgett, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+in+situ+transcription+and+splicing+in+large+human+genes.">Rates of in situ transcription and splicing in large human genes.</a> Nat Struct Mol Biol. 16, 1128-1133 (2009).</li><li>Brody, Y., Shav-Tal, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+transcription+in+real-time.">Visualizing transcription in real-time.</a> Cent Eur J Biol. 3, 11-18 (2008).</li><li>Lamond, A. I., Swedlow, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+II+transcription+in+living+color.">RNA polymerase II transcription in living color.</a> Nat Struct Mol Biol. 14, 788-790 (2007).</li><li>Darzacq, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+RNA+polymerase+II+transcription.">In vivo dynamics of RNA polymerase II transcription.</a> Nat Struct Mol Biol. 14, 796-806 (2007).</li><li>Boireau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+cycle+of+HIV-1+in+real-time+and+live+cells.">The transcriptional cycle of HIV-1 in real-time and live cells.</a> J Cell Biol. 179, 291-304 (2007).</li><li>Janicki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+silencing+to+gene+expression;+real-time+analysis+in+single+cells.">From silencing to gene expression; real-time analysis in single cells.</a> Cell. 116, 683-698 (2004).</li><li>Ben-Ari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+of+an+mRNA+in+space+and+time.">The life of an mRNA in space and time.</a> J Cell Sci. 123, 1761-1774 (2010).</li><li>Shav-Tal, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+single+mRNPs+in+nuclei+of+living+cells.">Dynamics of single mRNPs in nuclei of living cells.</a> Science. 304, 1797-1800 (2004).</li><li>Kremers, G. J., Gilbert, S. G., Cranfill, P. J., Davidson, M. W., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+at+a+glance.">Fluorescent proteins at a glance.</a> J. Cell Sci. 124, 157-160 (2011).</li><li>Sage, D., Neumann, F. R., Hediger, F., Gasser, S. M., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+tracking+of+individual+fluorescence+particles:+application+to+the+study+of+chromosome+dynamics.">Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics.</a> IEEE Trans Image Process. 14, 1372-1383 (2005).</li><li>Yunger, S., Rosenfeld, L., Garini, Y., Shav-Tal, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-allele+analysis+of+transcription+kinetics+in+living+mammalian+cells.">Single-allele analysis of transcription kinetics in living mammalian cells.</a> Nat Methods. 7, 631-633 (2010).</li><li>Brody, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+kinetics+of+RNA+polymerase+II+elongation+during+co-transcriptional+splicing.">The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.</a> PLoS Biol. 9, e1000573-e1000573 (2011).</li><li>Munoz, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+regulates+alternative+splicing+through+inhibition+of+RNA+polymerase+II+elongation.">DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation.</a> Cell. 137, 708-720 (2009).</li><li>Hieda, M., Winstanley, H., Maini, P., Iborra, F. J., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+populations+of+RNA+polymerase+II+in+living+mammalian+cells.">Different populations of RNA polymerase II in living mammalian cells.</a> Chromosome Res. 13, 135-144 (2005).</li><li>Kimura, H., Sugaya, K., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+cycle+of+RNA+polymerase+II+in+living+cells.">The transcription cycle of RNA polymerase II in living cells.</a> J Cell Biol. 159, 777-782 (2002).</li></ol>

用于测量活细胞聚合酶的动能活动的方法，可以分为两个部分。第一部分介绍的“湿”的过程，使mRNA转录的动力学数据测量和检索，而在第二部分中，数据是使用一个ODE模型分析。5一些研究现在已经利用这种方法提取在活细胞内5，6，8，12-14的转录动力学。这些研究之间的主要区别是FRAP恢复曲线转换成动能参数的方式。在许多情况下使用的数学模型。一个模型，描述了一个简单?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th></tr><tr><td>强力霉素</td><td>西格玛</td></tr><tr><td> FuGENE 6转染试剂</td><td>罗氏公司</td></tr></tbody></table>

measuring the kinetics of mrna transcription in single living cells

RNA聚合酶II（聚合酶II）的转录活性，是一个动态的过程， 因此测量在体内的转录过程中的动力学的重要性。聚合酶II动力学测量，利用生化或分子生物学方法。1-3近年来发展的新的可视化方法，它已成为可以遵循的转录发生在单个活细胞的实时4此中，我们介绍了如何执行在活细胞中特定基因的聚合酶II伸长动力学分析，5，6使用的细胞系，其中荧光标记一个特定的基因位点（DNA）的，其mRNA的产品，和最终的蛋白产物可在体内观察，它可以检测到感兴趣的基因转录的mRNA的实际，7，8的mRNA荧光标记使用标记的mRNA在体内，mRNA转录的3&#39;UTR含有24 MS2的茎环MS2的系统重复，提供YFP - MS2外壳蛋白标签的mRNA转录的高度特异性结合位点（ 九 ）监测转录的动力学我们使用漂白（FRAP）方法后，荧光恢复。通过在转录现场漂白新生成绩单YFP - MS2的标签，然后按照这种信号随时间的恢复，我们取得新的mRNA的合成率。，换句话说，YFP - MS2的荧光恢复，反映了一代人新的MS2在新生的成绩单和荧光自由YFP - MS2的分子，从周围的核质进入其约束力的茎环。 FRAP恢复曲线，然后使用一系列微分方程的数学机械化形式化的模型，以检索转录的动力学参数进行分析。

Watch this Scientific Journal Video about Measuring the Kinetics of mRNA Transcription in Single Living Cells at JoVE.com

Measuring the Kinetics of mRNA Transcription in Single Living Cells

在活细胞中的特定基因上RNA聚合酶II转录的动力学测量。感兴趣的基因转录的mRNA荧光标记，并使用荧光漂白后（FRAP）在体内转录伸长动力学得到恢复。

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Research

JoVE Journal

Biology

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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 ...

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 ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...