Name: simple and robust in vivo and in vitro approach for studying virus assembly
Uploaded: 2012-03-01T12:00:00
Description: Watch this Scientific Journal Video about Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly at JoVE.com

笔者想在注射浸润的发展和在体外装配检测实验室的几名成员，感谢他们的宝贵建议。这项工作是由从美国加州大学的资助。部分支持这项研究是由国家科学基金会（CBET-1144237）的资助。

1。植物材料<ol><li> 烟草在包壳实验中使用的植物应该是在4叶期（约3-4周老厂）。 </li></ol> 2。植物注射浸润细胞病毒基因组的功能部件的交付和表达<ol><li> 第1天：农杆菌菌株（如高血压105或GV 3101）窝藏pCass-BMV RNA，BMV RNA 2和BMV RNA 3 1,2种植为辅，卡那霉素50毫克/毫升LB琼脂板（选择农杆菌pCASS载体）和利福平100mg/ml（选择）。孵化器在28°C正在开展为?...

<ol>
	<li>Annamalai, P., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-independent+expression+of+genome+components+and+capsid+protein+of+brome+mosaic+virus+in+planta:+a+functional+role+for+viral+replicase+in+RNA+packaging.">Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.</a> Virology. 338, 96-111 (2005).</li><li>Annamalai, P., Rofail, F., Demason, D. A., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+packaging+mechanism+in+positive-strand+RNA+viruses:+synchronized+coexpression+of+functional+multigenome+RNA+components+of+an+animal+and+a+plant+virus+in+Nicotiana+benthamiana+cells+by+agroinfiltration.">Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.</a> J. Virol. 82, 1484-1495 (2008).</li><li>Annamalai, P., Rao, A. L., Foster, N., Johansen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Virology Protocols. 451, 251-264 (2008).</li><li>Lane, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bromoviruses.">The bromoviruses.</a> Adv. Virus Res. 19, 151-220 (1974).</li><li>Choi, Y. G., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+studies+on+bromovirus+capsid+protein.+VII.+Selective+packaging+on+BMV+RNA4+by+specific+N-terminal+arginine+residuals.">Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.</a> Virology. 275, 207-217 (2000).</li><li>Sambrrok, J., Russel, 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. , (2001).</li><li>Dreher, T. W., Rao, A. L., Hall, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+in+vivo+of+mutant+brome+mosaic+virus+RNAs+defective+in+aminoacylation.">Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation.</a> J. Mol. Biol. 206, 425-438 (1989).</li><li>Jung, B., Rao, A. L., Anvari, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+nano-constructs+composed+of+genome-depleted+brome+mosaic+virus+doped+with+a+near+infrared+chromophore+for+potential+biomedical+applications.">Optical nano-constructs composed of genome-depleted brome mosaic virus doped with a near infrared chromophore for potential biomedical applications.</a> A.C.S. Nano. 5, 1243-1252 (2011).</li><li>Voinnet, O., Lederer, C., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+movement+protein+prevents+spread+of+the+gene+silencing+signal+in+Nicotiana+benthamiana.">A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.</a> Cell. 103, 157-167 (2000).</li><li>Wroblewski, T., Tomczak, A., Michelmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Agrobacterium-mediated+transient+assays+of+gene+expression+in+lettuce,+tomato+and+Arabidopsis.">Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis.</a> Plant Biotechnol. J. 3, 259-273 (2005).</li></ol>

这里介绍的注射浸润方法，可以广泛适用于广泛的植物病毒。这种方法的一个共同特征是同步交付多个agroconstruct相同的细胞，通常经常使用的植物病毒机械接种相关的一个主要缺点。 在体内和体外装配使用雀麦花叶病毒模型的研究，可以有效地进行，由以下几个小技巧。（i）对于N 的成功渗透本生离开，没有水的浸润前24小时的植物;（二）农杆菌悬浮培养扩散到细胞?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MES钠盐</td><td> Sigma-Aldrich公司</td><td> M2993 </td></tr><tr><td>吲哚青绿</td><td> Sigma-Aldrich公司</td><td> I2633 </td></tr><tr><td>贝克曼超速离心机</td><td>贝克曼</td><td>型号：L8-70M </td></tr><tr><td> centricon-100列</td><td> Millipore公司</td><td> YM-100 </td></tr><tr><td>分光光度计</td><td>卡里50，瓦里安公司</td><td>零件编号 10068900 </td></tr><tr><td>分光光度计</td><td>乔宾-Yvon公司的Fluorolog 3。 </td><td>零件编号FL3-21 </td></tr></tbody></table>

simple and robust in vivo and in vitro approach for studying virus assembly

在与正义RNA基因致病人类，动物和植物，后代包壳进入成熟和稳定的病毒粒子的病毒是在一个特定的主机的基数在建立感染阶段。因此，包壳的研究解密的信息，调节病毒组装的机制，形成传染性病毒颗粒的诀窍。在制定遏制病毒的传播和疾病控制的新方法，这些信息是至关重要的。病毒包壳，可以在体内和体外研究。基因在体内的包壳，是一个高度管制的选择性的过程，涉及大分子的相互作用和亚条块分割。因此，研究领导剖析事件，其中包括在体内的病毒包壳，将提供的基本知识，了解如何病毒增殖和组装。最近在体外包壳已开发为在IMA生物医学领域的研究ging和治疗中的应用。非笼罩植物病毒纳入企业在体外带负电荷的外来物质的包壳中遥遥领先。雀麦草花叶病毒（BMV），非笼罩多元的RNA病毒致病的植物，已被用来作为一个模型系统研究基因在体内和体外包装。在烟草植物的包壳检测， 农杆菌介导的瞬时表达，是指作为注射浸润，高效和强大的技术相同的单元是一个用于同步多个组件的交付和表达。在这种方法中， 农杆菌携带二进制进行的desiredviral基因cDNA的载体细胞悬浮到间隙withina的使用没有超过1毫升一次性注射器（针）的复杂的叶渗透。的T-DNA转移到植物细胞中的DNA插入这个过程的结果;插入瞬时仍然在细胞核，然后由主机聚合酶II转录，导致瞬时表达。由此产生的转录（封顶，加尾），然后出口到细胞质中的翻译。约24至48小时的潜伏期后，渗透叶子的部分，可以为microscopyor生化分析采样。注射浸润允许细胞大量（数百到数千），同时要转。在体外包壳，BMV纯化病毒衣壳蛋白分离，对分离缓冲含有钙，RNA和非游离病毒颗粒通过离心去除氯透析。贫基因衣壳蛋白亚基重组所需的病毒基因组元件或如吲哚菁染料的非病毒成分。

Watch this Scientific Journal Video about Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly at JoVE.com

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

同步提供多个病毒成分对植物细胞通过一种简单，高效和可靠的方法<em&gt;农杆菌</em&gt;介导的瞬时表达描述。这种方法是适合就读复制，包壳其次<em&gt;在体外</em&gt;到非病毒部分基因组的重组耗尽适用于生物医学应用光学病毒鬼。

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Research

JoVE Journal

Immunology and Infection

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

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