支持这项工作是由NIAID的（R01AI074951，U54AI057168）和宾夕法尼亚大学基因组到SC前沿研究所资助，由美国国立卫生研究院T32HG000046到TSM;从美国国立卫生研究院T32GM07229和T32AI007324劳资关系组。

第1部分：在384孔板的RNAi <ol><li>筛选所用试剂的标准化是必不可少的。我们测试个别地段施耐德的媒体，胎牛血清，和染色试剂。然后，我们分装和使用整个屏幕的同一地段。 </li><li>对于任何检测，这是最好的测试几种不同的细胞系，以确定哪些是最适合你的生物读出的。我们通常使用的S2 - DRSC线。 </li><li> 果蝇细胞成长在25 ° C在完成施耐德的媒体。 <ol class="lalpha"><li>施耐德公司的媒体</li><li> 10％热灭活胎牛血清</li><li> 1X大号-谷氨酰胺</li><li> 1X笔/链球菌抗生素</li></ol></li><li> 果蝇细胞半贴壁和代无胰蛋白酶关闭保温瓶的吹打细胞和稀释1:10到一个新瓶中的细胞每四天。细胞应在实验阶段日志。 </li><li>通过使用多孔板所有液体自动液体处理板到板和每天的日常变异最小化。我们使用一个WellMate（矩阵）。 </li><li>准备WellMate管。 <ol class="lalpha"><li>喷雾用70％乙醇的发动机罩和WellMate。 </li><li>从包装和喷雾用70％乙醇管。 </li><li>在配药WellMate的位置插入管卡带。 </li><li>总理管25毫升70％乙醇。 </li><li>在乙醇中消毒15分钟，然后用另一乙醇25 ml总理管。 </li><li>通过吸用50毫升无菌PBS冲洗管道。 </li></ol></li><li>打跑瓶和计数细胞。颗粒细胞，使你有25％以上（300xg，5分钟）。根据测定的长度，每孔接种细胞的数量必须优化。进行自动图像分析时，一个单一的单层无细胞聚集，是理想的图像分割。 </li><li> 1.7x10 6细胞/ ml颗粒细胞重悬于无血清施耐德的媒体。 </li><li>我们执行的250ng/well浓度与市售的dsRNA库预分装在384孔板的屏幕。每块板的几口井空的控件，这是手动添加。我们用双链RNA的荧光素酶作为阴性对照。我们使用对主题（dIAP）双链RNA作为一个强大的RNAi控制。击倒，这种戏剧性的细胞死亡的抗凋亡因子结果，是一个快速直观的方式来评估敲质量。最后，我们使用一个双链RNA对病毒的记者，在这种情况下的β- galacosidase，作为阳性对照，验证，我们可以使用我们的检测读出抑制病毒的感染。如果发现成板的dsRNA在细胞播种，分装成的井，以便更容易可视化的角落1μL的双链RNA时，再离心到井底300xg，（1分）的双链RNA，然后加入细胞。 </li><li>使用WellMate添加以上的准备，以每孔10μL，细胞。 </li><li>自旋离心300xg，1分钟到板的细胞。 </li><li>在25 °彗星孵化器孵育45分钟。 </li><li>加入20微升完成施耐德的媒体和离心机300xg，1分钟。 </li><li>放置在加湿的特百惠容器板内衬水浸泡过的纸巾。 384卷，是小;因此，蒸发生物学，这反过来又可以导致在边缘井读出的artifactual变化上可以有很大的影响。为了克服这个问题，板应保持在高湿度。 </li><li>孵育3天，让强大的击倒。有没有必要改变媒体或在这段时间内打开加湿容器。 </li></ol>第2部分：与牛痘病毒感染<ol><li>消毒多渠道Quatricide 15分钟的吸气，然后用清水冲洗70％的乙醇。 </li><li>准备WellMate如上所述。 </li><li>完整的媒体1:5稀释血清中的自由媒体，准备用2％FBS感染施耐德的媒体。 </li><li>通过0.8微米的注射器过滤去除细胞碎片，或用纯化的病毒过滤器的原油病毒库存。 </li><li>稀释牛痘病毒在2％血清施耐德的媒体获得的感染复数（MOI）2。 </li><li>删除PBS WellMate油管，并用稀释的病毒的主要油管，直到无气泡。 </li><li>使用消毒多渠道吸引器和真空歧管，轻轻地清除双链RNA 治疗的果蝇细胞板的媒体。 </li><li>免除到使用的WellMate板，每孔30μL病毒。 </li><li>在300xg离心板1分钟。 </li><li>消毒WellMate管。总理与25毫升10％bleaCH，等待10分钟，并与另外25毫升10％的漂白粉总理。 </li><li>其次是50毫升70％乙醇50 mL水总理油管。返回其包装的油管。 </li><li>返回加湿特百惠容器板。在25 ° C培养48小时。 </li></ol>第3部分：染色板<ol><li>固定板15微升4％甲醛/ PBS 10分钟。液体被删除，使用多渠道吸引器和真空多方面的每一步。一旦板块已经固定，无菌设备和试剂已不再需要。 </li><li>删除修复和使用WellMate加30μLPBST（PBS + 0.1％的Triton - X）。孵育10分钟，然后重复。操作WellMate如上所述，除管不需要进行消毒。 </li><li>为了检测牛痘感染，我们使用了β-半乳糖苷酶的记者，早/晚痘苗病毒启动子驱动。 </li><li> 10分钟，取出块中的液体，并培育的细胞（2％BSA PBST）。 </li><li>稀块中的主要抗体，除去液体，并加入15μL抗体，每口井重复使用多通道移液器（矩阵）。 </li><li>离心板（300xg，1分钟），盖有一个明确的贴纸，4℃过夜孵育。 </li><li>删除的主要抗体，并使用WellMate PBST 3次，每次10分钟，洗井。 </li><li>稀释荧光标记二抗（FITC）和核染色块中，并加入15μL井使用多渠道重复移液器。 </li><li>孵育板在室温下1小时，由轻保护。 </li><li> PBST 3次，每次10分钟使用的WellMate洗井。 </li><li>密封板明确的贴纸，盖，并将其存储在4 ° C至三个星期。 </li></ol>第4部分：成像和图像分析<ol><li>使用20X放大倍率的自动显微镜图像板。 </li><li>共核（DAPI）和感染的细胞（FITC）捕捉的图像。优化的曝光时间分别为每个通道，以最大限度地提高动态范围。 </li><li> ž堆栈的图像，除了间隔1微米，即预期的焦点平面的上方和下方的飞机跨越微调自动对焦功能。一旦最佳焦平面的位置，相应地调整自动对焦设置。 </li><li>整个板块图像，捕捉的Hoechst通道（DAPI）和病毒的通道（FITC）每口井至少有三个图像。选择从几个不同的区域内，以及土地，以捕捉到的图像代表以及作为一个整体。 </li><li>使用MetaXpress软件写日记来分割图像，并使用Count核模块计算出的细胞为核染色（细胞总数）和感染（FITC）为每个板块阳性的总数。 </li><li>计算％的感染（100 * FITC阳性/总细胞）和日志转换。 </li><li>要确定候选人，我们使用基于每块板的中位数和四分位距的一个功能强大的Z评分。这种方法是对异常和非对称数据，从而提供了更大的功率确定，即使弱或中度命中10。对于每个板块，中位数是从数转换的数据中减去。然后把结果值分为四分位距（75 个百分位值和25 个百分位之间的差额）的0.74倍。 0.74的因素是用来近似标准正态分布。 </li><li>强大的Z分数是衡量标准偏差距离该板块的中位数。例如，一个强大的Z - score模型2％以及感染是从板块中位数或P &lt;0.05〜2标准偏差。 </li><li>分析细胞数量，以确定细胞毒性候选人。核计数，计算功能强大的Z评分。与一个强大的Z &lt;重复屏幕-2候选人被认为是细胞毒性。 </li><li>井无细胞毒性％&gt; 2或&lt;重复屏幕-2（P &lt;0.0025）感染的一个强大的Z得分，被认为是从主屏幕上的潜在候选人。 </li><li>积极的候选人是使用独立的试剂验证。 dsRNAs对从其他地区的mRNA的兴趣的基因产生和测试以上。 </li><li>可以考虑进一步研究这些基因的两个独立的dsRNAs有相同的表型。 </li></ol>第5部分：代表性的图像和解释的感染率<img src="/files/ftp_upload/2137/2137fig1.jpg" alt="图1" /> 图1未感染井不显示任何染色牛痘病毒蛋白，而感染以及有代表性的包含痘苗病毒表达β-半乳糖苷酶（βgal）蛋白作为免疫荧光显微镜测量，染色阳性的细胞。病毒=绿色，细胞核=蓝色。 <img src="/files/ftp_upload/2137/2137fig2.jpg" alt="图2" /> 图2。自动图像分析软件量化感染在每个图像使用由用户设置的参数。在一个形象代表，总数计数的细胞核和细胞表达病毒抗原的数量，大小和强度高于当地的背景染色染色的基础上。 <img src="/files/ftp_upload/2137/2137fig3.jpg" alt="图3" /> 图3。击倒线程（dIAP）作为阳性对照为强劲的RNAi靶基因的枯竭。这种抗凋亡因子击倒导致戏剧性的细胞死亡。核=蓝色。 <img src="/files/ftp_upload/2137/2137fig4.jpg" alt="图4" /> 图4。击倒的萤光素酶作为阴性对照为双链RNA对感染的治疗效果。荧光素酶的消耗相对左未经处理的细胞没有感染的效果。病毒=绿色，细胞核=蓝色。 <img src="/files/ftp_upload/2137/2137fig5.jpg" alt="图5" /> 图5。βgal蛋白质击倒作为一个在减少感染的阳性对照，自检测使用βgal的蛋白水平，作为一个读感染。消耗在感染细胞的百分比下降的βgal结果。病毒=绿色，细胞核=蓝色。 <img src="/files/ftp_upload/2137/2137fig6.jpg" alt="图6" /> 图6。击倒Rab5细胞因子在感染细胞的百分比下降的结果。由于Rab5是已知参与内吞作用，这一因素可能有助于牛痘病毒进入。这代表了从屏幕上的例子离群。病毒=绿色，细胞核=蓝色。

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

在果蝇的全基因组的RNAi筛选提供了一个强大和有效的方法，研究病毒感染的细胞成分。一些哺乳动物病毒感染的果蝇细胞，它可以用来识别主机的固有免疫反应的组成部分和主机病毒的复制，可能代表新的治疗目标所需的蛋白质因子。执行屏幕前，必须精心优化检测类型的细胞，细胞数量和感染程度，，必须得到很好的控制，包括正反两方面的病毒和细胞的目标11。之间的阳性和阴性对照，以确保足够的分离是很重要的事先筛选，最大限度地提高检测的动态范围。已执行的画面后，考生可以分成功能类别，以确定哪个主机机制有助于感染。对于哺乳动物，如牛痘病毒，通过RNAi的哺乳动物同源的贡献应确定二次分析的一部分。两者合计，这些强大的筛选方法将让我们获得洞察到其中的病毒与宿主细胞相互作用的复杂机制。

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<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
<td>&nbsp;</td>
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<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
<td>&nbsp;</td>
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<td>Penicillin/streptomycin (pen/strep)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
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<td>Fetal Bovine Serum (FBS)</td>
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<td>Corning</td>
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<td>Clear seals</td>
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<td>Denville</td>
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<td>Well Mate</td>
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<td>B2261</td>
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<td>Fluorescently-labeled secondary antibodies</td>
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<td>Invitrogen</td>
<td>&nbsp;</td>
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<td>ImageXpressMicro automated microscope</td>
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<td>MetaXpress image analysis software</td>
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rnai screening for host factors involved in vaccinia virus infection using drosophila cells

病毒性病原体是一个重大的公众健康构成威胁;病毒不仅能引起人类疾病的天然流行病，但他们在生物恐怖袭击的潜在用途也是一个问题。一个更好地了解细胞因子的影响感染，将有利于发展急需的疗法。 RNA干扰（RNAi）再加上几个生物的完整基因组测序技术的最新进展，导致全基因组，细胞为基础的损失的功能屏幕优化。果蝇细胞由于基因组规模屏幕特别适合在这个系统中1 RNAi的易用性和效率。重要的是，多种病毒可以感染果蝇细胞，包括哺乳动物病毒的医疗和农业的重要性2,3,4数量。前RNAi技术在果蝇中发现屏幕上有主机，是病毒感染的各个步骤，包括入境，翻译和RNA 的复制5所必需的因素。此外，许多在果蝇细胞培养的病毒复制所需的细胞因子也限制在与这些病毒4,6,7,8，9感染的人体细胞。因此，增选在病毒感染的宿主因素的识别抗病毒疗法的新目标。在这里，我们提出了一种高通量RNAi筛选，以确定参与病毒感染的细胞因子， 使用牛痘病毒为例的广义协议。

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

在病毒感染的新型主机所涉及的因素可以识别通过细胞为基础的的全基因组功能RNAi筛选的损失。一<em&gt;果蝇</em&gt;细胞培养模型的易用性和效率的RNAi由于这种方法特别适合。在这里，我们展示了这种技术使用<em&gt;牛痘</em&gt;病毒为例。

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

rnai-screening-for-host-factors-involved-vaccinia-virus-infection

Research

JoVE Journal

Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

9.4K 次观看.  University of Pennsylvania. 在病毒感染的新型主机所涉及的因素可以识别通过细胞为基础的的全基因组功能RNAi筛选的损失。一果蝇细胞培养模型的易用性和效率的RNAi由于这种方法特别适合。在这里，我们展示了这种技术使用牛痘病毒为例。

视频: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

科研

免疫与感染

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...