这项工作是由美国国立卫生研究院研究院（NIH R01 AI46435）RMRCE（NIH AI065357）支持。

以下协议的书面基于辛德毕斯病毒MRE16的一个安非他明类兴奋剂的生产和使用。安非他明类兴奋剂的目的是表达GFP在蚊子埃及伊蚊 。目前已为安非他明类兴奋剂的（见表1）设计的alphaviruses（辛德毕斯病毒，基孔肯雅病毒，O&#39;nyong倪翁病毒，西部马脑炎病毒）的cDNA感染性克隆。为了达到最佳效果，确保主管细菌（Stratagene公司/安捷伦科技），以避免不必要的病毒基因的重组和损失，如细菌质粒DNA扩增。所有的感染性克隆质粒有“病毒的cDNA全长基因组RNA的转录和一个独特的限制性内切酶在3的径流转录结束”结束噬菌体T7或SP6的启动子在眼前5。对于每一个安非他明类兴奋剂，用户必须使用相应的噬菌体DNA依赖的RNA聚合酶和独特的限制性内切酶在体外转录病毒的RNA基因组。 1。从cDNA克隆的感染性RNA的生成。 <ol><li>线性化的质粒XhoI位限制性内切酶20单位100μL反应（p5&#39;dsMRE16 / GFP）的感染性克隆的5μg。 2 - 3H在37 ° C </li><li>净化线性DNA用Qiagen公司QIAprep自旋小量制备试剂盒备用净化协议，从旋转列使用50μL无RNase水洗脱DNA。质粒浓度应约为1.0微克/μL。 </li><li>在一个无RNase的0.2 mL PCR管中，设置50μL逆转录反应，每个协议使用Ambion公司MAXIscript套件如下。 <ul><li> 22.5μL无RNase DH 2 O <ul><li> 5.0μL，线性DNA模板（1.0μg/μL的） </li><li> 2.5μL，10mm的三磷酸腺苷</li><li> 2.5μL，10MM的CTP </li><li> 2.5μL，10mm的双绞线</li><li> 2.5μL，1毫米的GTP </li><li> 2.5μL10MM第模拟（米7个G（5&#39;）PPP（5）G） </li><li> 5.0μL10X转录缓冲液</li><li> 5.0μLT7或SP6的聚合酶混合</li></ul></li><li> 50.0μl的</li></ul></li><li>转录反应1小时在37 ° C。孵育后，应立即使用反应，BHK - 21细胞电。电的BHK - 21细胞的制备应开始孵化过程中的转录反应。 </li></ol> 2。病毒感染RNA的生成<ol><li> Trypsinize两个70-80％汇合150厘米2（T - 150）容量瓶中BHK - 21细胞每安非他明类兴奋剂。 </li><li>所有细胞转移至15 mL锥形离心管。 </li><li>在2000转离心沉淀细胞，10分钟，4℃在传统的台式离心机。 </li><li>在无菌PBS悬浮细胞无MG + +和Ca + +的。重复步骤2.3和2.4，直至细胞已用PBS洗3次。 </li><li>悬浮颗粒细胞加入0.5 mL的MEM含10％胎牛血清。 </li><li>用90μL含10％胎牛血清的MEM稀释10μL的细胞，并依靠一个hemacytometer。如果必要，稀释细胞2.5-12.5 × 10 7细胞/毫升纪念含10％胎牛血清。 </li><li>一个冰冷的0.2厘米电比色皿中，加400μL细胞悬液20μL逆转录反应。从比色皿擦拭凝结。 </li><li>脉冲试管两次：450V，1200Ω，150μF。我们使用的电细胞机械臂，哈佛器械型号ECM630。 </li><li> 25厘米2组织培养5毫升的MEM含10％胎牛血清的烧瓶中放入试管的全部内容。 </li><li> 37C（S）用5％的CO 2孵育烧瓶。 </li><li>每天使用适当的过滤器设置（如GFP的过滤器倒置荧光显微镜：激发波长为460-490纳米，发射波长为510-550纳米;或DsRed的过滤器：激发波长为460-560纳米，发射波长监控感染=&gt; 590纳米）。 </li><li>当荧光表明感染是融合和/或CPE是可见整个烧瓶，烧瓶中收集的内容，添加30％FBS，必要时，并储存于-80等分° C。如果需要的话，细胞裂解液可能被清除的离心（2000转，10分钟，4℃）胎牛血清除了之前。 </li><li>通道病毒至少一次通过新的细胞增加为随后的血液喂养检测病毒滴度烧瓶。 </li></ol> （3） 每个OS感染蚊<ol><li>混合在一起的血液（通常购买去fibrinated羊血）和细胞培养步骤2.13来自病毒悬液。血粉最终病毒滴度通常应〜1 × 10 7斑形成单位（PFU）/毫升高效的肠病毒感染的蚊子。为了刺激蚊子大吃，腺苷5&#39; -三磷酸腺苷（ATP）可以添加到终浓度为0.02M。 </li><li>蚊子可能水和糖剥夺前感染，激发他们从人工接驳有效血饲料。先前被剥夺的时间应建立，以减低死亡率。时间将取决于蚊种，湿度insectary等作为一个起点，除去饲料糖24小时前水12h。该协议使用37℃的热水循环水套式的玻璃feeders17。猪的肠膜（即彻底清洗肠衣在大多数杂货店）是放置在馈线和餐进入会议厅吸管口。可针对不同类型的载体的各种图案，膜和协议。 </li><li>蚊子排序只选择那些充血的蚊子。充血蚊子转移到一个纸箱顶部与蝉翼纱和蚊子都孵育28℃，相对湿度为75-80％，直至处理GFP的表达。 </li></ol> 4。胸腔内接种蚊胸腔内接种感染节肢动物的替代方法。这种方法是使用时不需要通过传播肠。 （下面描述的方法，但技术不是在这种视觉的实验显示）。 <ol><li>一个小蚊子（5-10）被吸入到塑料控股管的控股笼。控股的密封管将被放置在4 ° C下15分钟冷却的蚊子。 </li><li> 2 5蚊子会从管到一个玻璃培养皿菜搁在冰上洒。放置在培养皿的盖子时，在不使用或如果蚊子开始走动。在蚊子的处理，必须小心计数，并保持跟踪被操纵的蚊子数量。由于蚊子都洒到寒意表，总数将被记录在实验室计数器。 </li><li>准备使用Nanoject特定的玻璃和针拉马熔点毛细管针。 </li><li> 69 NL接种交货安装Nanoject第二微量每制造商的指示。绘图时进针的接种，使针头不被损坏，必须小心。 </li><li>使用解剖显微镜，蚊子的胸部插入针和激活接种Nanoject。必须小心时，接种针，使不完全渗透的蚊子和退出的另一边，在随后死亡的蚊子蚊子。检查每个蚊子，以确保从针接种前进入。 </li><li>接种后，蚊子仍然针头刺穿，放置在一个抱着纸箱的纸，通过小孔的一方，是插在所有其他时间用棉花或一个软木塞。 </li><li>当蚊子接种放入箱（每箱约50），仔细磁带到位软木的蚊子，以防止意外释放。放置在insectary进一步孵化前的安全举行，在28 ° C和80％相对湿度下的bin纸箱。 </li></ol> 5。监测感染的蚊子<ol><li>抱着纸箱的纸放置在4℃，约15分钟到固定的蚊子。 </li><li>将一个小的蚊子数量（一次5-10）使用荧光显微镜适应寒意表。 </li><li>审查和排序基础上存在或没有荧光的蚊子。此外，荧光强度可能被用来作为代理感染的定量测量。蚊子，如肠，唾液腺组织，脂肪体解剖和荧光监测。 </li><li>如果合适，纸箱返回选定的蚊子，继续受感染的蚊子的潜伏期。 </li></ol> 6。传输检测（强制流涎演示病毒传播） <ol><li>剥夺24小时前喂蚊子糖源。 </li><li>删除的腿和翅膀</li><li> 50μL的毛细管已加热，拉，并在一端剪断，添加3-5μLCargille B型浸油或10％血清的蔗糖溶液。 </li><li>插入管的长鼻。如果使用油，唾液飞沫将散发出的长鼻。可应用于胸部刺激pilocarbine解决方案在PBS的1％和0.1％吐温80​​μL。 </li><li> 60-90分钟后，消除蚊子和病毒分离的存储，如果需要的话。 </li><li>从管中取出离心管中含有100μL20％FBS - PBS的解决方案。 </li><li>无菌过滤器通过注射器0.2微米过滤器的接种，然后用过滤的接种感染培养细胞。 </li></ol> 生物安全注意：此协议介绍一个产生，识别和监测受感染的蚊子的方法。根据你的设备（即寒意表，遏制设施等）和国际生物伦理委员会的批准，你可能是有限的，以检查他们只取出后，翅膀和腿，以防止逃逸。 SINV为基础的安非他明类兴奋剂的，可以在一个BSL2环境中生成和使用。使用安非他明类兴奋剂的基孔肯雅病毒或西部马脑炎病毒，如其他alphaviruses需要BSL3设施。 7。代表性的成果使用安非他明类兴奋剂5&#39;dsMRE16 / GFP（GFP）的一个标记基因的表达概述如图1所示。 GFP表达在BHK - 21和C6/36细胞（ 白纹伊蚊 ）是在感染后的细胞5&#39;dsMRE16 / GFP的病毒多重感染在0.01倍，如图2所示。图3是甲病毒和安非他明类兴奋剂病毒感染时，该病毒是通过感染血粉交付的蚊子的概述。图4显示了一个血粉〜10 7 PFU /安非他明类兴奋剂病毒，其次是口腔感染埃及伊蚊毫升的准备。全身感染的蚊子和使用epifluorescence显微镜（图5）选定的身体部位，在感染后10天的看法。绿色荧光蛋白基因的检测，并在受感染的蚊子表示每个荧光标记基因（图6）与安非他明类兴奋剂5&#39;dsMRE16病毒感染肠DsRed的。传输实验使用5&#39;dsMRE16 / GFP安非他明类兴奋剂（图7）病毒感染的蚊子。 表1。目前，安非他明类兴奋剂的设计，在蚊子的基因表达。 <table border="1"><tbody><tr><td>&nbsp; 甲病毒 </td><td> 安非他明类兴奋剂 </td><td> 蚊子 </td><td> 参考 </td></tr><tr><td>辛德毕斯病毒</td><td> TE / 3&#39;2J和TE / 5&#39;2J </td><td> 埃及伊蚊 </td><td> 12 </td></tr><tr><td></td><td></td><td> Ochlerotatus triseriatus </td><td> 13 </td></tr><tr><td></td><td> 3&#39;dsMRE16和 5&#39;dsMRE16 </td><td> 埃及伊蚊 </td><td> 5 </td></tr><tr><td></td><td></td><td> 三带喙库蚊 </td><td> 5 </td></tr><tr><td></td><td> 5&#39;dsTR339 </td><td> A. AEG ypti </td><td> 2 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>基孔肯雅病毒</td><td> 3&#39;dsCHIKV和 5&#39;dsCHIKV </td><td> 埃及斑蚊/ A伊蚊 </td><td> 20 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> O&#39;nyong倪翁病毒</td><td> 5&#39;dsONNV </td><td> 按蚊 </td><td> 1,9 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>西部马脑炎病毒</td><td> 5&#39;dsWEEV。麦克米兰</td><td> 致倦库蚊tarsalis </td><td> Stauft 等，未发表</td></tr></tbody></table> 表2。使用安非他明类兴奋剂的蚊子基因表达的优点/缺点。 <table border="1"><tbody><tr><td> 优点： </td></tr><tr><td>快速生产高滴度的病毒</td></tr><tr><td>广泛的宿主范围（SINV） </td></tr><tr><td>高RNA的复制率</td></tr><tr><td>高的转基因表达水平</td></tr><tr><td>操纵基因的相对缓和</td></tr><tr><td>稳定，持久性基因的表达昆虫</td></tr><tr><td>在昆虫中最小的病理</td></tr><tr><td></td></tr><tr><td> 缺点： </td></tr><tr><td>短期在脊椎动物细胞中的表达模式</td></tr><tr><td>脊椎动物细胞的细胞病变效应强</td></tr><tr><td>基因的大小限制（&lt;1KB，理想情况下） </td></tr><tr><td>有些alphaviruses需要BSL3遏制</td></tr></tbody></table><img src="/files/ftp_upload/2363/2363fig1.jpg" alt="图1" /> 图1：安非他明类兴奋剂的BHK - 21 p5&#39;dsMRE / GFP SINV表达GFP细胞的病毒生产概述。安非他明类兴奋剂的基因组RNA是体外转录后，电穿孔进入BHK - 21细胞病毒是救出。然后，可以使用安非他明类兴奋剂病毒感染的蚊子。小额赠款=亚基因组启动子。三安非他明类兴奋剂的RNA物种转录的CELLS，基因组，亚基1和亚基2的RNA。 C（衣壳蛋白），E2和E1糖蛋白与安非他明类兴奋剂的基因组组装生成传染性病毒。 <img src="/files/ftp_upload/2363/2363fig2.jpg" alt="图2" /> 图2：与SINV 5&#39;dsMRE16 / GFP病毒感染后的细胞表达绿色荧光蛋白（C6/36）蚊子。 <img src="/files/ftp_upload/2363/2363fig3.jpg" alt="图3" /> 图3：安非他明类兴奋剂病毒感染导致病毒传染的蚊子的概述。 <img src="/files/ftp_upload/2363/2363fig4.jpg" alt="图4" /> 图4：安非他明类兴奋剂SINV 5&#39;dsMRE16感染由蚊子感染血粉。 <img src="/files/ftp_upload/2363/2363fig5.jpg" alt="图5" /> 图5：安非他明类兴奋剂SINV 5&#39;dsMRE16 / GFP感染在感染后10天。绿色荧光蛋白的全身检测表明，病毒躲过了肠，并分发给中学组织。图也显示选定的身体部位，也是一个传播感染的指示GFP的表达。 <img src="/files/ftp_upload/2363/2363fig6.jpg" alt="图6" /> 图6：安非他明类兴奋剂SINV 5&#39;dsMRE16 / GFP和5&#39;dsMRE16 / DsRed的感染，在感染后的特定时间的肠。肠通常有不同灶摄取一个血粉含贯穿在稍后的时间后感染后肠病毒，传播后早期感染。 <img src="/files/ftp_upload/2363/2363fig7.jpg" alt="图7" /> 图7：早在感染后8天的安非他明类兴奋剂5&#39;dsMRE16 /绿色荧光蛋白在蚊子的唾液检测。检测的目的是显示的安非他明类兴奋剂的病毒，并显示5&#39;dsMRE16 / GFP病毒可以稳定表达的GFP整个外在潜伏期蚊子传播的。左侧面板中显示了毛细管垂涎欲滴的蚊子，中央面板显示C6/36细胞，在感染后3天1天，右侧面板用唾液感染。

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+chloramphenicol+acetyltransferase+in+Aedes+albopictus+(C6/36)+cells+and+Aedes+triseriatus+mosquitoes+using+a+double+subgenomic+recombinant+Sindbis+virus.">The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.</a> Insect Biochem. Mol. Biol. 24, 39-39 (1994).</li><li>Olson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Sindbis+virus+expression+system+that+efficiently+expresses+green+fluorescent+protein+in+midguts+of+Aedes+aegypti+following+per+os+infection.">Development of a Sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of Aedes aegypti following per os infection.</a> Insect Mol. Biol. 9, 57-57 (2000).</li><li>Parikh, G. R., Oliver, J. D., Bartholomay, L. C., C, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+haemocyte+tropism+for+an+arbovirus.">A haemocyte tropism for an arbovirus.</a> J. Gen. Virol. 90, 292-292 (2009).</li><li>Pierro, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+orally+infectious+Sindbis+virus+transducing+system+that+efficiently+disseminates+and+expresses+green+fluorescent+protein+in+Aedes+aegypti.">Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti.</a> Insect Mol. Biol. 12, 107-107 (2003).</li><li>Rutledge, L. C. W. a. r. d., A, R., Gould, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+feeding+response+of+mosquitoes+to+nutritive+solutions+in+a+new+membrane+feeder.">Studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder.</a> Mosq. News. 24, 407-407 (1964).</li><li>Sanders, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sindbis+virus+induces+transport+processes+and+alters+expression+of+innate+immunity+pathway+genes+in+the+midgut+of+the+disease+vector+Aedes+aegypti.">Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector Aedes aegypti.</a> Insect Biochem. Mol. Biol. 35, 1293-1293 (2005).</li><li>Uhlirova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Sindbis+virus-mediated+RNA+interference+to+demonstrate+a+conserved+role+of+Broad-Complex+in+insect+metamorphosis.">Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 15607-15607 (2003).</li><li>Vanlandingham, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+double+subgenomic+chikungunya+virus+infectious+clone+to+express+heterologous+genes+in+Aedes+aegypti+mosquitoes.">Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosquitoes.</a> Insect Biochem. Mol. Biol. 35, 1162-1162 (2005).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inducing+or+inhibiting+apoptosis+on+Sindbis+virus+replication+in+mosquito+cells.">Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells.</a> J. Gen. Virol. 89, 2651-2651 (2008).</li></ol>

这里介绍的方法，使研究人员能够按照感染甲病毒在蚊子细胞培养和成年雌蚊。使用安非他明类兴奋剂病毒的优势和缺点，总结在表2。一个感染性克隆安非他明类兴奋剂的基因可以被操纵，以表达任何印度政府，并已被用来表达单链抗体，RNA干扰抑制器，内源性基因的反义RNA的目标，促凋亡蛋白。如果记者不使用，那么这种方法的成像部分是不相关的。然而，许多相同的协议是安非他明类兴奋剂病毒救援，传播，和蚊子感染所必需的。有最大规模的转基因插入到甲病毒转导系统时的局限性。大小的限制取决于用于生成的安非他明类兴奋剂的父母应变而有所不同，但通常约1KB，虽然较大，如萤火虫荧光素酶的报告基因已兴建蚊子使用安非他明类兴奋剂的使用。研究实验室的病毒学背景温和的金额应能使用安非他明类兴奋剂的这些协议。一些研究实验室已经做了，因此使用SINV为基础的安非他明类兴奋剂的。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>QIAprep Spin Miniprep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>MAXIscript Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AB1312</td>
<td>Be sure to use a kit containing the appropriate polymerase for your construct.</td>
</tr>
<tr>
<td>Cap Analogue (m7G(5')ppp(5')G)</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM8050</td>
<td>&nbsp;</td>
<tr>
<td>Nanoject II nanoliter injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>Electro Cell Manipulator</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>ECM 630</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

alphavirus transducing system:  tools for visualizing infection in mosquito vectors

甲病毒转导系统（ATSS）表达基因（GOI），在感染过程中的重要工具。 ATSS来自蚊子传播的RNA病毒的cDNA克隆（ 属甲病毒; 家庭 Togaviridae） 。 甲病毒属包含约30种不同的蚊子传播的病毒种。 Alphaviruses包膜病毒含有单链RNA的基因组（〜11.7 KB）。 Alphaviruses抄写encapsidation病毒的基因组和成熟所需的病毒结构蛋白编码的一个亚基的mRNA。 Alphaviruses通常高度在脊椎动物细胞裂解，但持续感染与最小的细胞病理学容易受到蚊子的细胞。这些特性使之成为优秀的工具，以免蚊虫的基因表达。在使用中最常见的ATSS来自辛德毕斯病毒（SINV）。 SINV广泛的物种向性允许对感染的昆虫，禽流感，并哺乳动物cells8。然而，ATSS已被来自其他alphaviruses以及9,10,20。外源基因表达是可以通过插入一个额外的病毒亚基因组RNA起始位点或启动。其中一个外源基因序列定位的病毒结构基因5&#39;ATSS是用于稳定在昆虫蛋白的表达。 ATSS，其中一个基因序列定位3“的结构基因，是用来触发RNAi和沉默，在昆虫的基因的表达。 ATSS已被证明是有价值的工具，对于理解向量的病原体相互作用，病毒复制的分子细节和维护3,4,11,19,21传染性周期。特别是荧光灯和发光记者表达已跟踪5,14-16,18载体和病毒传播的病毒感染。此外，载体的免疫反应已经用两个SINV设计，以表达绿色荧光蛋白 2,9株。 在这里，我们提出了一个SINV生产方法从cDNA感染性克隆一个含有荧光记者（GFP）。传染病，全长RNA转录从线性的cDNA克隆。传染病RNA引入宽松的靶细胞，通过电。转染的细胞产生感染性病毒颗粒，表示印尼政府。收获病毒传染给蚊子，这里描述的，或其他宿主物种（此处未显示）。矢量能力评估肠以外的荧光检测或监测病毒的传播 7 。使用一种荧光记者，印尼政府允许病毒传播的方便估计整个细胞培养，确定感染，在暴露的蚊子，从传播病毒的蚊子传播的速度，并提供一个矢量能力的快速指标。

Watch this Scientific Journal Video about Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors at JoVE.com

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

使用甲病毒转导系统，以表达荧光记者在体外和成蚊的方法进行了阐述。这种技术可适应任何利益代替或除了向记者表达的蛋白质。

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

alphavirus-transducing-system-tools-for-visualizing-infection

Research

JoVE Journal

Immunology and Infection

14.7K 次观看.  Colorado State University. 使用甲病毒转导系统，以表达荧光记者在体外和成蚊的方法进行了阐述。这种技术可适应任何利益代替或除了向记者表达的蛋白质。

视频: Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

科研

免疫与感染

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

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 ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...