我们感谢我们的实验室的成员，富有成果的讨论和技术援助。这个视频演示支持研究和兽医病理学部的副院长，兽医卫生科学中心，美国俄克拉荷马州立大学。 Ministerio DE Ciencia发送Innovación，西班牙（项目BFU2008-01244/BMC），中船重工壁间项目PA1002451樱资助这项研究是由食品动物研究的Walter R. Sitlington赋主席猴王，CVHS 2009 RAC补助，OAES动物健康基金和美国农业部，国家研究计划的竞争性赠款，号2007-04613。

1。产生双链RNA。 <ol><li>综合寡核苷酸引物含有T7启动子序列在体外转录合成双链RNA（例如，为草原革栎 subolesin使用寡核苷酸引物D8AAT75： TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT 5&#39; - 3&#39;和D8DVT73：5&#39; - TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG - 3&#39;）。 </li><li>使用10 pmol每个寡核苷酸引物和蜱总RNA的1-10吴用RT - PCR扩增的靶基因。 </li><li>净化的PCR产物。 </li><li>使用8μL纯化的PCR产物合成的dsRNA。 </li><li>量化法的双链RNA。 </li></ol> 2。注射用双链RNA的刻度。 2.1。准备注射用的刻度。 <ol><li>晃动在每个解决方案，在50 mL一次性离心管，细网线在管顶屏幕保留蜱通过调迁的解决方案，首先，洗了一系列的解决方案中的蜱。洗涤蜱的解决方案的顺序是自来水，3％的双氧水，蒸馏水，70％的乙醇，用蒸馏水洗涤洗涤。 </li><li>干纸巾吸干蜱。 </li><li>数到20至50组的蜱，取决于实验，放置在一个1.25盎司塑料杯蜱从每个组配备一个紧密的盖子，并与实验组编号的标签。 </li></ol> 2.2。勾选注射团队。 RNA干扰队由三人组成：（1）一个人，谁的立场上双粘性胶带贴一张红色的牙科用蜡每个刻度，（2）的人注射后监控蜱蜱（3）一个人注射蜱，呼吸的CO 2来激活它们并计数到实验组编号标记杯的生活蜱。所有团队成员都必须戴一次性手套。 2.3。安置注射蜱。 <ol><li>使用杜蒙罚款钳捕获蜱和地方双重粘性胶带贴3“× 6”红牙科用蜡表腹侧。蜱密切定位在5蜱组。 </li><li>放置胶带超过5蜱的口器，以进一步抑制他们的小片，但同时离开身体最暴露，使注射过程中，可以通过蜱喷射器（图1）观察。 </li></ol> 2.4。注射的刻度。 <ol><li>蜱将被注入在较低的外骨骼腹面右象限。 </li><li>首先，刺穿一个洞在使用装有一个半“，29号针头（图2a）一个Monoject胰岛素注射器的外骨骼。 </li><li>蜱立即注入0.2-0.5μL的双链RNA的解决方案-使用一个特制的汉密尔顿注射器1英寸，33号针头与45 °斜面点（5 × 10 5 × 10 的11％微升分子）（图2b） 。针应放入剔腔，以确保安置和保留的双链RNA。一些液体可能逃走，从注射部位（图2c）。应注意不要过度注入蜱，这将导致损失的血淋巴，并可能导致死亡的Tick。 </li><li>清洁完成后，各实验组注射汉密尔顿注射器。在另一个实验组使用。首先填充注射器从一个烧杯中含有3％的双氧水，然后驱逐到物的容器，并重复15次。填充注射器从一个包含无菌水的烧杯中，然后驱逐到物的容器，并重复15次。小心不要弯曲汉密尔顿注射器的柱塞，柱塞，因为，如果弯曲不会顺利地移动和响应，以注射蜱需要温柔的触摸。 </li></ol> 2.5。治疗注射后的刻度。 <ol><li>立即拿起双胶带注射用细镊子蜱和放置在一个塑料的回收容器（约6“× 6”，并用胶带环抱，以防止蜱逃生）。蜱将简要注射后无效，但应该很快就会开始抓取周围的菜。 </li><li>呼吸二氧化碳到蜱后立即放置在回收容器中，他们帮助激活蜱2。一旦蜱爬行活跃，注射伤口迅速愈合，他们将最有可能生存。 </li><li>计数的蜱，根据各实验组的数量和放置在一个标签的塑料杯，他们紧密装上盖子。更换蜱应注射，以取代任何，任何未来的实验组注射前死亡。 </li></ol> 2.6。蜱举行。 <ol><li>放置在一个湿度室蜱（12小时灯：12小时黑暗的光照在22-25 ° C和95％相对humidity），并持有1天。 </li><li>广场蜱，蜱喂养细胞，每一个实验组，粘到了羊，并让他们与同等数量的男性或女性uninjected蜱（取其性是不注射）饲料。雌蜱饲料饱食，从羊后10天喂养或控制女性删除那些已下车的收集和权衡的主机。 </li><li>放置在纸箱中的蜱，直到完成产卵和湿度室举行。加权评估组中的所有蜱的卵块产卵。 </li></ol> 2.7。 RNA干扰的刻度表型分析后。 <ol><li>评估喂奶后打勾确定蜱幸存，剔重，产卵和卵生育的数量，表型。然而，其他分析可能是执行上的靶基因和研究目标而定。 </li></ol> 3。通过RT - PCR分析，以确认基因沉默。 <ol><li>剖析喂奶后注入控制和双链RNA注射组个人蜱唾液腺和胆量。 </li><li>从个别组织样本中提取总RNA。 </li><li>实时RT - PCR分析个别组织的目标基因的转录和RNA水平正常化，对蜱16S rRNA的使用genNorm法（ddCT Bio - Rad公司iQ5标准版，2.0版中实现的方法）。 </li><li>解离曲线运行时的反应，以确保只有一个扩增形成的结束，在相同的温度范围内为每个样品一致的扩增产物变性。 </li><li>比较mRNA水平（标准化的Ct值）控制喷油和双链RNA注射蜱使用学生的t检验（P = 0.05）之间。 </li></ol> 4。代表性的成果： 该协议所述均使用在许多不同的ixodid打勾物种在我们实验室的RNAi（见表1）。量的dsRNA注入蜱随蜱的大小;较大的蜱种，可以容纳体积较大。负控制蜱应无关的双链RNA注射。几个dsRNAs如subolesin 14-19,22-25,27-32,34和β-肌动蛋白20,21可作为阳性对照。请注意，重要的是要洗的治疗，以避免混合的dsRNA解决方案之间的注射器。如果协议是正确的，应获得不少于5％的死亡率从24小时后注射过程。在蜱的基因敲除后的一个典型的表型是为了打勾的保护性抗原注射双链RNA池屏幕面板的刻度图3所示。 <table border="1"><tbody><tr><td> 蜱种 </td><td> 双链RNA注射 </td><td> 参考文献 </td></tr><tr><td> 肩突硬蜱 </td><td> cDNA文库，subolesin，肌动蛋白，核苷酸，NF - kB的，akirin </td><td> 21，22，29，30 </td></tr><tr><td> 草原革栎 </td><td> subolesin，消费税，泛素，vATPase，硒蛋白M和W2A，造血干/祖细胞的蛋白，肌动蛋白的蛋白酶体26S亚基，ferritin1，varisin，akirin </td><td> 15，19，22，24，26，30-32 </td></tr><tr><td> 草原革marginatus </td><td> subolesin </td><td> 22 </td></tr><tr><td> 蜱americanum </td><td> cDNA文库，subolesin，akirin </td><td> 17，22，30 </td></tr><tr><td> 蜱hebraeum </td><td> subolesin，voraxin </td><td> 28 </td></tr><tr><td> 镰形扇头sanguineus </td><td> Rs86，subolesin </td><td> 22，23 </td></tr><tr><td> 镰形扇头microplus </td><td>商品及服务税，泛素，硒，Bm86，Bm91，subolesin，胃肠道，GIII，EF1a，flagelliform蚕丝蛋白，血管性血友病 因素</td><td> 16，18，25，27 </td></tr><tr><td> 镰形扇头annulatus </td><td>泛，subolesin，EF1a，GIII </td><td> 16 </td></tr></tbody></table> 表1。勾选其中的RNAi协议已使用的物种。 <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="图1" /> 图1。蜱的位置，坚持一张红色的牙科用蜡粘在双磁带，腹侧起来。蜱被放置在5个一组，之后，一个是放置在为了进一步巩固，同时允许注射器注射过程中观察身体的蜱蜱的口器，小片胶带。 <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="图2" /> 注射过程包括：（一）刺入较低的刻度外骨骼与胰岛素SY右象限图2 。灵厄，以创建一个注射部位装有29号针头，（b）立即在这个场地使用33号针头（三）最​​有可能会导致一些蜱血淋巴漏/汉密尔顿注射器注射的双链RNA流体。 <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="图3" /> 图3。蜱六个组RNA干扰在其中被用来在蜱americanum蜱保护性抗原的屏幕面板。积极subolesin RNAi的控制和负无关的双链RNA控制相比，可以看出在蜱的表型变化。在这个实验中，各组蜱死亡率，重量，和产卵的RNAi效果进行统计分析。

<ol>
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Acarol. 46, 17-28 (2008).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+the+role+of+tick+subolesin+in+gene+expression.">Evidence of the role of tick subolesin in gene expression.</a> BMC Genomics. 9, 372-372 (2008).</li><li>Smith, 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+impact+of+RNA+interference+of+the+subolesin+and+voraxin+genes+in+male+Amblyomma+hebraeum+(Acari:+Ixodidae)+on+female+engorgement+and+oviposition.">The impact of RNA interference of the subolesin and voraxin genes in male Amblyomma hebraeum (Acari: Ixodidae) on female engorgement and oviposition.</a> Exp. Appl. Acarol. 47, 71-86 (2009).</li><li>Galindo, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+subolesin+is+an+ortholog+of+the+akirins+described+in+insects+and+vertebrates.">Tick subolesin is an ortholog of the akirins described in insects and vertebrates.</a> Dev. Comp. Immunol. 33, 612-617 (2009).</li><li>Canales, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+immunogenicity+of+the+mosquito+ortholog+of+the+tick+protective+antigen,+subolesin.">Conservation and immunogenicity of the mosquito ortholog of the tick protective antigen, subolesin.</a> Parasitol. Res. 105, 97-111 (2009).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+genes+involved+in+Anaplasma+marginale-tick+interactions+affects+the+pathogen+developmental+cycle+in+Dermacentor+variabilis.">Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.</a> BMC Dev. Biol. 9, 42-42 (2009).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subolesin+expression+in+response+to+pathogen+infection+in+ticks.">Subolesin expression in response to pathogen infection in ticks.</a> BMC Immunol. 11, 7-7 (2010).</li><li>Karim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+tool:+gene+silencing+in+Ixodes+scapularis+eggs+and+nymphs+by+electroporated+dsRNA.">Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.</a> BMC Biotechnol. 10, 1-1 (2010).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA.">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tick-pathogen+interface+for+novel+control+strategies.">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li><li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila.">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li></ol>

虽然其他方法已被用于RNAi蜱14，33，注射双链RNA这里描述的是最广泛使用的unfed（表1）和美联储蜱 16,25,34 。 RNAi技术已被证明是一个有价值的工具，蜱基因功能的研究，蜱病原体接口的特性， 甄别和14,35蜱保护性抗原的特性。特别是，RNA干扰已成为最有价值的35蜱功能分析工具。 方法论，RNA干扰可能会演变为更有效的方法，可能允许在大量的个人基因敲除。双链RNA诱导的RNA干扰机制蜱应进一步完善，有助于更好地了解和利用这种在35,36这个物种的遗传方式。蜱RNAi的脱靶效应的程度也是一个重要的问题需要得到充分解决 14,27 。最后，RNAi技术将最有可能提供全面的贡献蜱基因调控的研究和系统生物学和蜱病原体的接口，并可能有一个疫苗的开发上的影响，控制蜱侵袭和蜱传播病原体的传播。

<!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>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

蜱是野生和家养动物和人类的预留hematophagous体外寄生虫，被认为是全球第二蚊子人类疾病和最重要的载体媒介影响养牛业的全球 2 。蜱是在子类蜱螨亚纲，亚目，为了Parasitiformes Ixodida分类和分布在世界各地从北极到热带地区3。尽管努力控制蜱侵袭，这些体外寄生虫仍然是一个严重的问题，为人类和动物健康4,5。 RNA干扰（RNAi）是一个基于核酸的反向遗传学方法，涉及到基因表达的干扰，以确定一个代谢途径上的基因的功能，其效果。小干扰RNAs（siRNAs）是由双链RNA（dsRNA）的结果在一个强有力的序列特异性降解细胞质的mRNA含有相同的序列的dsRNA触发7-9发起的RNAi途径效应分子。由双链RNA发起的转录后基因沉默机制已经在所有真核生物中发现的研究迄今为止，和RNAi已迅速开发各种生物作为功能基因组学研究和其他应用程序10的工具。 RNA干扰已成为使用最广泛的基因沉默技术在蜱和其他生物的基因操纵的替代方法不可用，或者是不可靠的5,11。直到最近的RNAi 12,13应用受到了限制遗传特性的刻度。在短的时间内，RNA干扰已被提供，它已被证明是一个有价值的工具，为研究打勾基因功能，蜱病原体接口的特性和14蜱保护性抗原的筛选和表征。在这里，用于RNAi的dsRNA注入unfed蜱的方法是描述。它可能是从这个实验的方法获得的知识将有助于显着的基本生物系统的理解和疫苗的发展，控制蜱侵袭和防止蜱传播的病原体15-19传输。

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

unfed蜱的dsRNA注入一种RNA干扰（RNAi）的方法描述。 RNAi是使用最广泛的基因沉默在其他基因操纵方法的使用受到限制，蜱技术。

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Research

JoVE Journal

Immunology and Infection

18.0K 次观看.  Oklahoma State University. unfed蜱的dsRNA注入一种RNA干扰（RNAi）的方法描述。 RNAi是使用最广泛的基因沉默在其他基因操纵方法的使用受到限制，蜱技术。

视频: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...