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）将绑定到法律咨询及田土转易处重复和标记的DNA 。 YFP - MS2的蛋白质结合到MS2的重复（茎环）标签的mRNA。该基因的产物是CFP -肌动蛋白，因此诱导的基因会导致CFP -标记的肌动蛋白细胞骨架的外观。是转基因构建成一个U2OS四面体的稳定细胞系和其表达的是春节的转录控制下。 1。的细胞电镀和转染： <ol><li>测量前48小时，种子1〜2 × 10 5细胞在玻璃见底组织培养皿（35毫米培养皿菜与一个14毫米的玻璃底微孔（厚度为0.16-0.19毫米;猫号P35G - 1.5 - 14 - C MatTek，阿什兰，马萨诸塞州））。添加中等​​2毫升细胞，以达到50-80％汇合。 </li><li>实验前24小时，瞬时转染的细胞结构，表达的荧光标记物标记的DNA和mRNA的。使用FuGENE6转染试剂（Roche公司）。 所用的构造： <ol type="a"><li> YFP - MS2，免入息审查贷款计划将标签中的mRNA转录MS2的茎环。 YFP - MS2蛋白的核定位序列（NLS）的提供核项目序列。 </li><li>可选 - CFP - LACI或RFP - LACI将绑定在5个基因的“LACO重复，将标签的基因位点。在追踪期间FRAP实验的基因标记基因位点助攻，但是没有必要的。 </li></ol></li><li>孵育至少12小时的细胞，获得良好的瞬态转蛋白（见注）的表达。 </li><li> 6 - 12小时的实验前增加1.5微克/毫升强力霉素（Sigma公司）春节诱导CFP -肌动蛋白基因的激活细胞。 </li></ol>注： *重要的是没有达到很高的转染蛋白的表达水平，以免掩盖获得相对背景信号转录的网站，特别是信号。表达水平可以控制：a）调整之间的转染和实验的时间; b）改变启动子的强度（如巨细胞病毒是比L30发起人强）; C）加入核出口信号（NES） YFP - MS2，免入息审查贷款计划的蛋白质，这将有助于散布在整个细胞YFP的信号。 *用于测量荧光最好耐光整个长收购，因此，绿色/黄色发光的荧光团比红色荧光团。不同的荧光团的资料，可以发现文献10。 *确定不同的光渠道不流血通过彼此。使用含有与每个单独的标记标记标本幻灯片，每张幻灯片测量两个通道的强度，然后计算渗出。 2。 FRAP： 任何能够获取图像和执行漂白用激光束显微镜，可以进行实验。通常情况下，FRAP实验用激光扫描共聚焦显微镜（CLSM）。然而，因为在我们的例子的实验是比较长（15-30分钟），和我们下面的细胞核内的一个小的轨迹，重要的是能够保持焦点在整个实验的基因位点/转录网站。这就要求在3个维度（4D）随着时间的推移快速成像，例如采集速率是很难获得使用扫描共聚焦显微镜。因此，我们使用一个3D - FRAP还原系统（Photometrics）建立一个宽领域的奥林巴斯IX81显微镜配备EM - CCD（QUANT - EM，罗珀）（63X计划APO，1.4 NA），能够获得高速的图像（最多30个图像/秒）。该系统具有405纳米和491纳米激光器，可与摄像头，使样品的特定区域的利益率（ROI）漂白同步。该显微镜是配备了一个lambda DG - 4光源（沙特）和一个XY，Z阶段（前），允许在Z轴的快速，准确的成像。所有的设备集成和MetaMorph（Molecular Devices公司）所控制。在37 ° C，5％CO 2进行活细胞的实验使用的是活细胞室系统（东海）。 <ol><li>程序：实验前半小时，打开加热装置，显微镜和激光。的实验期间MENT的温度需要保持不变，否则可能会导致在漫长的采集时间的焦点变化。激光功率必须是稳定的，一致的输出功率。 </li><li>同步与摄像头的激光： <ol><li>搜索中不包含细胞（使用DIC的）的板面积。 </li><li>使用相同的光通道将用于实验所用的。在这里，我们用491 nm激光为YFP的通道。 </li><li>激光点的图像的中间。 </li><li>使用同步“按钮（宏）。 </li><li>尝试在该领​​域的不同点，以确保激光和摄像机同步。 </li></ol></li><li>选择一个很好的候选细胞实验。虽然我们与稳定的细胞系工作，我们发现，并非所有的细胞也有类似的表达水平。很好的候选人，将细胞：A）表现出明显的YFP - MS2 -标签弥漫YFP - MS2的背景之上的转录网站，B）有足够的自由YFP - MS2蛋白在细胞内，以便能够完全恢复后，漂白（例如，在图1B - 比较两个单元格的左上角单元格是不是一个很好的候选人进行漂白）。 </li><li>选择合适的测量条件。 FRAP还原过程需要许多图像的获取，因此重要的是要合理的曝光时间，并使用尽可能低的光之间的平衡。高光水平可能会影响细胞因光毒性，并会导致漂白。它是建议保持成像参数不同的实验之间的常数。 细胞成像YFP的通道在150毫秒的曝光时间，在CFP CFP - LACI（基因组位点）的检测通道。 CFP - LACI标签位点，使该基因的检测，即使YFP - MS2 mRNA信号光漂白（可选）。对于每个收购，7 Z型片每350纳米。活跃​​转录的网站是使用491 nm激光漂白。 </li><li>执行完整的实验，但没有褪色。 FRAP实验措施转录网站需要返回到稳定状态的时间。首先，我们要验证活跃转录的网站是处于稳定状态的功能和稳定。因此，我们执行一个完整的FRAP的实验，但没有漂白，以确保测量强度的转录网站在实验过程中的时间框架不会改变。我们用这个实验中也测试通过测量成像系列所造成的总漂白的成像条件。作为一个经验法则，完整的电影光漂白，不应该超过30％的初始强度。这可以使用在细胞核中的相同点从过去的形象，除以第一张图像（比例应小于0.7较大）的强度比来衡量。 </li><li> FRAP：YFP - MS2的信号活跃转录的网站是使用491 nm激光漂白。 6漂白前的图像采集。漂白后的图像采集了3个时间频率序列：每3秒15的图像，每6秒和26（或45长期实验）的图像，每30秒15的图像。在获取图像的频率变化，允许在最高强度的变化（初步复苏），并接近尾声图像信号的变化不太突出时代获得更多图片。 </li></ol>注：这个实验，从“常规”，FRAP不同： *网站在转录的mRNA的FRAP措施YFP - MS2蛋白新生的mRNA结合，而典型的FRAP实验测量细胞在不同的蛋白质的流动性。因此，有没有兴趣在测量YFP - MS2蛋白扩散池。当漂白和第一次获得的图像之间的时间也比较长，很可能无视这个免费YFP - MS2的蛋白质的扩散池。 YFP - MS2的信号恢复*由于相对较长（15-30分钟），重要的是要保持重点转录网站。这就是为什么在每一个时间点收购一系列的Z -片。 3。正常化的FRAP数据： 4D FRAP数据分析使用免费的图像分析软件ImageJ（国立卫生研究院，马里兰州贝塞斯达; <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> ）。以下是宏通过ImageJ软件进行分析，但自编写的宏，当然可以生成。 <ol><li> Z轴堆叠最大预计在每一个时间点，随着时间的推移，作进一步的分析2D电影。 </li><li>转录的网站是在每帧跟踪和平均强度测量使用点跟踪工具。11中的数据保存在Excel工作表，作进一步的分析。 </li><li>对于每一个时间点，在其他地区的利息率（ROI）的平均强度测量如下（使用时间序列分析的ImageJ插件）：A）背面地面是从一个细胞= B（T）之外的投资回报率。二）投资回报率在核质，但尽可能远离漂白网站可能= C（T）。 C）转录网站= S（T）（现场跟踪测量）。 </li><li>在成像过程中造成的漂白正确的数据：减去所有其他测量的背景，然后正确的漂白。 SC（T）是纠正在时间t的力度转录网站</li></ol> 式1： <img src="/files/ftp_upload/2898/2898eq1.jpg" alt="公式1" /><ol start="5"><li>规范化数据：首先计算漂白前的图像，这表明在稳定状态（= &lt;Sc (pre-bleach)&gt;）的转录网站强度平均。 </li></ol>然后正常化的数据： 式2 <img src="/files/ftp_upload/2898/2898eq2.jpg" alt="公式（2）" />至少有10实验数据归。计算实验的平均值和标准差（STD）的，在每一个时间点（图1C）的错误酒吧。 注： 在FRAP分析YFP - MS2蛋白扩散忽视，因为自由nucleoplasmic YFP - MS2的扩散速度非常快，而绑定的YFP - MS2具有高亲和力的mRNA，并没有重新连接到的转录网站。这就是为什么漂白后的第一个形象，应在分析前删除。 4。使用了一个数学模型的数据分析： 为了获取这种数据集的动力学参数，我们用最简单的描述模型，该模型可以适应这种数据。这一特定模式的完整说明，可以读取这里 5 。该模型包含下面的公式描述图2A计划： <img src="/files/ftp_upload/2898/2898eq3.jpg" alt="公式3" /> <img src="/files/ftp_upload/2898/2898eq4.jpg" alt="公式4" /> 模型的参数，它是可以计算的伸长率： <img src="/files/ftp_upload/2898/2898eq5.jpg" alt="公式（5）" /> 我们分析使用伯克利麦当娜（ <a href="http://www.berkeleymadonna.com">http://www.berkeleymadonna.com</a> ）的曲线，如以前为蓝本5，但其他选项可用（MATLAB，虚拟细胞，COPASI等）。数据拟合模型和速率常数分别提取。我们在伯克利分校的麦当娜附加代码。为了以最适合的数据，我们计算了稳定状态，然后适合归归模型的实验数据。 5。代表性的成果： 我们光漂白使用激光功率高的网站在积极转录标记基​​因的信号，并遵循随着时间的推移YFP - MS2的信号恢复。转录现场的荧光信号由稳态动力学的mRNA的合成，以及从该网站公布的表达。未经漂白转录网站强度随着时间的推移测量，给出了一个“恒”信号（图1C，红点），这意味着，这种基因是目前在一个相对稳定的状态。这意味着，新转录的mRNA积累的信号等于信号，离开时，一个mRNA转录网站发布。当光漂白，这个系统将继续在稳定状态，但现在可以测量它的信号来恢复所需的时间。因此，荧光的恢复主要取决于聚合酶的延伸特性。 3D FRAP系统允许收购的全3D FRAP恢复期间的核体积，实验过程中丢失了，从而没有信号。从数据计算的动力学参数，归一化曲线拟合，基于微分方程的动力学模型描述伸长5使用一个简单的ODE模型的优点是，它可以适合只有少量的参数。该模型的初始步骤（切入点），伸长率，分别负责合成新的MS2的结合位点的过程。最终的步骤（退出点）是mRNA进入核质的释放。该模型的基础上恢复曲线，动力学解决的两个组件组成，一个快速和一个慢。快速的组成部分，是指伸长率，而速度较慢的组件建模聚合酶暂停期间伸长。两个聚合酶状态为蓝本，作为一个随机过渡暂停伸长。我们从这个模型优化的差分方程，取得了3.3 KB /分钟的伸长速度与随机过渡到一个较慢的合成率（累计时间为2.5分钟暂停）。模型表明T暂停受影响的只有11％的聚合酶进入伸长伸长过渡。 <table border="1"><tbody><tr><td> K掉</td><td> 0.0112 </td><td></td></tr><tr><td> K pause_in </td><td> 0.0015 </td><td></td></tr><tr><td> K pause_out </td><td> 0.0067 </td><td></td></tr><tr><td>伸长率时间</td><td> （K掉+ K pause_out）-1 </td><td> 56秒</td></tr><tr><td>暂停时间</td><td> （K pause_out）-1 </td><td> 2.5分钟</td></tr><tr><td>暂停的可能性</td><td> （K pause_in）/（K pause_in + K掉） </td><td> 11％ </td></tr></tbody></table> 表1模型的参数表中的结果。 <img src="/files/ftp_upload/2898/2898fig1.jpg" alt="图1" /> 图1。 （一）实验细胞系统， 用于体内基因表达。 CFP -肌动蛋白基因构建的示意图。 5&#39; -端包含了一系列的256个法律咨询及田土转易处 ，也必然RFP - LACI（红色）和标记基因整合的网站重复。从最小的CMV启动子的转录诱导是通过反向四环素转录激活因子（rtTA或四面体在）的结合春节反应元件（TRES）DOX存在。转录的mRNA含有CFP -β-肌动蛋白的编码序列，和24 MS2的重复，YFP - MS2融合蛋白的约束。在3&#39; -末端，一个兔β-珠蛋白外显子 - 内含子 - 外显子模块的一部分，其次是一个卵裂多聚腺苷酸信号。该计划还介绍了伸长率检测显示荧光的MS2蛋白（黄色）连接到新转录的RNA聚合酶II转录的mRNA（b）在活跃转录的网站（YFP - MS2，右手细胞）是光漂白和荧光恢复被跟踪随着时间的推移（帧的底部）。我们测得的荧光强度漂白剂（中间线）和非漂白（底线）积极转录。注意左手的细胞是不适合一个漂白实验（C）CFP -肌动蛋白基因的转录网站上进行的实验测量。在蓝色经济复苏的YFP - MS2的FRAP测量曲线。红色，处于稳定状态的转录网站YFP - MS2的信号来自同一实验的非漂白细胞。 <img src="/files/ftp_upload/2898/2898fig2.jpg" alt="图2" /> 图2。 （二）（a）计划的描述与转录现场YFP - MS2的分子标记的mRNA的入口和出口的动力学模型。YFP - MS2的FRAP数据拟合模拟（平均十个实验拟合模型） 。适合计算残差是在底部。

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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>

没有利益冲突的声明。

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 ...

measuring-the-kinetics-of-mrna-transcription-in-single-living-cells

Research

JoVE Journal

Biology

15.4K 次观看.  Bar-Ilan University. 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。

视频: Measuring the Kinetics of mRNA Transcription in Single Living Cells

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

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

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

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

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 ...

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

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

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

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent 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 is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

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

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

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

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

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

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

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

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

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) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

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 ...