Name: single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)
Uploaded: 2013-03-15T12:00:00
Description: Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

的的质粒pCG001表达Ubp1好心提供的罗汉贝克在约翰·科廷学校的医学研究。这项工作是由of Dimes的研究资助，2011财年3月，3月Dimes的瓦西里·奥康纳入门学术研究奖＃5-FY20和美国国家科学基金会职业奖0746796。

1。应变工程工作流程<ol><li>插入序列编码（一）的膜定位序列，（b）一种快速成熟的荧光蛋白和（c）的蛋白酶识别序列的N-末端和在帧与感兴趣的蛋白质（ 例如，一个转录因子）。我们采用针对性的TSR-金星记者2膜，并融合到的蛋白酶识别序列的泛素（Ub）表达λ噬菌体阻遏CI蛋白质分子的数量进行计数。我们如何构建的融合蛋白TSR-金星-泛素CI，CI编码区取代了野生型和将构建到<...

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	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, 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=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

CoTrAC方法可以推广到测量生产的常规的N-或C-末端的荧光蛋白融合的其它蛋白质，可能会破坏蛋白质的活性。 CoTrAC战略有三个独特的优势，目前的方法。首先，合作翻译融合确保为每个感兴趣的蛋白质分子的，一个分子中的荧光报道产生，允许实时蛋白质生产的准确计数。其次，膜 - 定位记者TSR-金星使荧光记者的单分子检测，从而能够检测在非常低的水平表达的蛋白质。第三和最重要的是，中的...

<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（可选） </td></tr><tr><td>琼脂糖（低熔融温度） </td><td>龙沙</td><td> 50100 </td><td></td></tr><tr><td> Milli-Q超纯水H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5×M9盐</td><td></td><td></td><td>以下配方9 </td></tr><tr><td> 20％葡萄糖</td><td></td><td></td><td></td></tr><tr><td> 硫酸镁 </td><td></td><td></td><td></td></tr><tr><td> 氯化钙 </td><td></td><td></td><td></td></tr><tr><td> 50×MEM氨基酸溶液</td><td> Invitrogen公司</td><td> 11130-051 </td><td></td></tr><tr><td>温度控制生长房间 舞台适配器</td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td>物镜加热器</td><td> Bioptechs </td><td>模型依赖于显微镜的物镜</td><td></td></tr><tr><td> Microaqueduct幻灯片</td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td>微盖玻片</td><td> VWR </td><td> 40CIR-1 </td><td>可能是困难的源，也可从Bioptechs </td></tr><tr><td>封面玻璃/滑动垫片</td><td> Bioptechs </td><td> FCS20.75毫米</td><td></td></tr><tr><td>荧光显微镜</td><td>各个</td><td>设置示例：相干伊诺308C氩离子激光，奥林巴斯IX-81显微镜，奥林巴斯PlanApo 100X NA 1.45目标，Metamorph软件</td><td>必须有激光激发下，自动化XYZ阶段，脚本和自动对焦成像的自动化软件，能够，能够resolvin光学g单荧光蛋白</td></tr><tr><td> EM-CCD相机</td><td>各个</td><td>设置示例：安道尔IXON DU-898 </td><td>必须具有足够低噪声检测到上述背景下的单一荧光蛋白</td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

我们描述的荧光显微镜法，共翻译激活由裂解（CoTrAC）图像与单分子的精确度不扰动的蛋白质的功能的情况下，在活细胞中的蛋白质分子的生产。此方法使得能够顺序，五分钟的时间窗期间在一个小区中产生的蛋白质分子的数目进行计数。它需要一个与激光激发功率密度为约0.5至1千瓦/厘米2，这是足够敏感的检测单一的荧光蛋白在活细胞中的分子的荧光显微镜。在此方法中所使用的荧光报道，由三部分组成：膜击靶序列，一个快速成熟的，黄色荧光蛋白和蛋白酶识别序列。记者感兴趣的蛋白质的N-末端的翻译融合。细胞生长在一个温度控制的显微镜载物台上。每五分钟后，细胞内的荧光分子成像（以及后来的合作unted通过分析荧光图像），并随后photobleached让只有新翻译的蛋白质在接下来的测量计数。 ，此方法产生的荧光图像，可以通过检测在每个图像中，将它们分配到单个细胞的荧光斑点，然后分配的细胞谱系的细胞进行分析。的数目计算出一个时间窗口内的一个给定的细胞产生的蛋白质的是，集成的斑点的荧光强度除以由单个荧光分子的平均强度。我们用这种方法测量的0-45的分子在单5分钟时间窗的范围中的表达水平。这种方法使我们能够测量噪声的λ阻遏CI的表达，并在系统生物学中有许多其他潜在的应用。

从CoTrAC跟踪生产的λ阻遏CI实验的典型结果示于图4和图5。在这个实验中，12个菌落成像以5分钟的间隔。在每个时间点，菌落第一自动对焦和集中的摄像区域内。接下来，中心/中心的位置，存储和一个亮场图像被收购。然后阶段易位〜0.5微米，从明场焦平面沿z轴移动的平面等分的细胞（无相位对比或微分干涉相差（DIC）的光学，半透明的对象可以被成像在稍外的聚焦平面<...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

我们描述的荧光显微镜法，共翻译激活由裂解（CoTrAC），对图像的生产与单分子的精确度不扰动的蛋白质的功能的情况下，在活细胞中的蛋白质分子。此方法已被用于按照随机表达动力学的一种转录因子，λ阻遏CI<sup&gt; 1</sup&gt;。

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Research

JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-Molecule Imaging of Nuclear Transport

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

科研

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

上皮钠通道（ENaC）的蛋白水解激活通过结合电流测量与检测裂解片段示范

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...