We would like to thank Dr. Flaminia Catteruccia and her research group (Harvard School of Public Health, Boston, MA) for providing our laboratory with the G3 colony used in this research and for the LacZ template DNA. We thank Adam Jenkins (Boston College, Chestnut Hill, MA) for his assistance in maintaining the insectary. The Biology Department of Boston College generously funded the research associated with the development of this technique.

1.合成和制备的dsRNA <ol><li>确定一个200-800碱基拦截区域（以产生相应的dsRNA）被预测为没有可识别的脱靶效应的感兴趣的基因内（ 例如 ，另一种基因中没有序列同源性≥18碱基对）和阴性对照（ 例如中，异源序列是不是目标昆虫基因组中存在，如大肠杆菌 lacZ基因（GenBank登录号基因ID:945006）。另外，使用阳性对照（ 例如 ，它产生一个容易观察的表型，如SRPN2 21,22 ，GenBank登录基因ID:1270169）所述dsRNA靶向SRPN2的序列在Michel 等人的定义（2005）21。 注:E-RNAi是一种开源生物信息学的资源，为这样的区域的鉴定和用于设计寡核苷酸引物23的过程是有用的。 </li><li>施行TANDARD PCR扩增（ 即 ，在使用Taq DNA聚合酶30-35个循环），使用基因组DNA或cDNA模板以获得由T7启动子序列侧翼的dsRNA合成的模板（5&#39;-TAATACGACTCACTATAGGG-3&#39;）中，用的dsRNA制剂进行并清理用市售的体外转录试剂盒按照制造商的说明。使用SRPN2 PCR扩增条件和作为所述的等人提出的引物信息。（2011）22。 </li><li>量化由紫外吸收光谱纯化的RNA扩增子的产量在260毫微米波长，并调整到在无RNase水所需的浓度（ 例如 ，3微克/微升）。 <ol><li>为低的RNA浓度进行故障排除，通过纺丝样品在室温下的真空离心机向下或通过冻干样品和在水较小体积重构减少液体体积。样品冻干所需要的时间将根据变化初始样品体积和双链RNA的浓度。 </li></ol></li><li>检查dsRNA的质量和长度上用1×TBE或TAE制备的2％琼脂糖凝胶缓冲液和用溴化乙锭（EtBr的）染色，与用于转录反应的模板DNA一起。的dsRNA会更慢比迁移模板DNA。质量和长度可以确保没有非特异性dsRNA的产品，分别比较产品与标准DNA标记，进行评估。 注意:乙锭是一种强效的诱变剂，应作相应处理。 注:在3微克/微升的dsRNA的浓度，0.5微升样品的比足够在溴化乙锭 - 染色的琼脂糖凝胶的可视化多。 </li><li>直到需要在-20℃储存的dsRNA。多个冷冻/解冻循环可能导致退化，所以等分应为大量的dsRNA来制备。 </li></ol> 2.准备快绿FCF染料（FGD）管<ol><li>快速稀释绿FCF染料从在无RNase水原液（≥85％染料含量）至0.1％（体积/体积）（工作液）。 </li><li>吸管1μl的染料至1.5 ml微量离心管的底部。 </li><li>在约3小时的65℃加热块地方管在室温下蒸发液体，然后发生管为至少30分钟，在使用前冷却。这种干燥的固体染料将在dsRNA的悬浮液重建。 </li></ol> 3.将注射针头<ol><li>拉用热针车夫到10-30微米的尖端直径硼硅玻璃针。拉设置对应于:加热器调整没有。 1 = 100，暖风调整没有。 2 = 70。 </li><li>为避免对针的细尖损伤，水平放置所有拉动针在培养皿上成形油灰的带状。 注意:毛细管针拉方法的更多的信息可以在进一步详细疟疾研究和参考试剂资源中心级M发现R4手册24。 </li></ol> 4.准备注射站<ol><li>收集所需材料:玻璃微细针被拉至精尖，微量，薄滤纸厚滤纸，培养皿，移液管，油漆刷和解剖光学显微镜。 </li><li>准备微量作为微量人工指示，和注射量设定为每脉冲所需的音量（ 例如 ，最高不超过69脉冲NL的）。 </li><li>在一个平台上，很容易在显微镜下操纵（ 例如 ，泡沫塑料管架的平坦的侧面），堆叠两个滤纸片在顶部薄滤纸，并与周围的边缘胶带固定。 </li><li>重悬10微升在不同的着色染料管各dsRNA的解决方案，并在冰上的地方。 </li></ol> 5.收集蛹注射液<ol><li>填充小60毫米×15毫米的培养皿用10毫升去离子水，并收集〜50浅蛹（杜里纳克从使用一次性塑料移液管的养虫托盘化蛹后的第一个24小时）。 </li><li>删除具有中等至暗角质层晒黑的蛹。 注意:一旦角质层开始晒黑，变得更加难以穿透角质层，并注射导致高得多的杀伤力。 </li></ol> 6.注射的dsRNA <ol><li>根据解剖显微镜，撅着注射针的远端有一对细镊子的。 </li><li>通过回填与矿物油的针（使用具有3英寸，30号针头的注射器），并且与微量排出多余的油制备的注射针。 </li><li>前填充注射针与dsRNA的最大数量，并在显微镜下喷射一个脉冲，以确保液体的分配。在没有液体被吸收和/或排出的情况下，检查针的远侧末端为任何堵塞，并确保针头牢固地固定在microinjecto河</li><li>挑蛹1-3，并将其放置到滤纸上。 </li><li>使用画笔，定位在与背角质层访问的滤纸的蛹，并使用画笔推的滤纸上，并吸收过量的水。 </li><li>稳定与画笔的尖端蛹，而在大约30°相对于蛹的背水面的角度针头插入的胸部和腹部之间的背角质层。注射应朝向蛹和针的后端被导向应的前至后方向上移动。 </li><li>注射3微克/微升dsRNA的解决方案两个脉冲（每个脉冲69 NL）进入淋巴，并为您的色彩在整个体内的分布。如果没有颜色标识，稍移注射针的位置从障碍物清除尖和重复液体输送。 </li><li>使用湿画笔轻轻地从针移动到蛹水养殖。 ŧ他蛹应该坚持在光接触画笔。 </li></ol> 7.注射后条件<ol><li>将培养皿注射蛹成蚊笼合适的气流（ 例如 ，网笼或网状盖容器）。蛹饲养条件应在27±3℃的温度始终保持75±5％的湿度和是一个光下:8小时:16的暗循环。 </li><li>制备10％（重量/体积）的葡萄糖溶液，并放置溶液饱和的棉球上蚊子笼网为成人喂养。 </li></ol> 8.评估击倒<ol><li>在所需的时间点上，在实验性的dsRNA注射昆虫评估表型，与对照组相比。 <ol><li>每日评估dsSRPN2和dsLacZ昆虫通过在-20℃冷却下成人为约2-3分钟，以2℃将它们转移到一个冷板和15X通过解剖显微镜的利用识别任何伪肿瘤形成放大倍率与明照明。评估后，返回成人杀虫条件（27±3℃，75±5％的湿度）。 注:在本协议所使用的实验和控制的dsRNA分别为dsSRPN2和dsLacZ。有适合于控制许多选项;然而，有人建议，阳性对照为哪些表型和/或表达是很容易可视化（ 例如 ，通过解剖显微镜法）和/或定量[ 例如 ，定量实时PCR（QRT-PCR），免疫印迹]应该使用学习通过Western印迹和定量RT-PCR检测了这种技术。SRPN2蛋白和转录物定量时，分别在Michel 等人。（2005）21中描述。 </li></ol></li></ol>

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<li>Luna, C., Wang, X., Huang, Y., Zhang, J., Zheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+four+Toll+related+genes+during+development+and+immune+responses+in+Anopheles+gambiae%5BTitle%5D)+AND+%22Insect+Biochem.+Molec.+Biol%22%5BJournal%5D)">Characterization of four Toll related genes during development and immune responses in Anopheles gambiae.</a> Insect Biochem. Molec. Biol. 32, 1171-1179 (2002).</li>
<li>Tauszig, S., Jouanguy, E., Hoffmann, J. A., Imler, J. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-related+receptors+and+the+control+of+antimicrobial+peptide+expression+in+Drosophila%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Toll-related receptors and the control of antimicrobial peptide expression in Drosophila.</a> Proc. Natl. Acad. Sci. 97 (19), 10520-10525 (2000).</li>
<li>Tryselius, Y., Samakovlis, C., Kimbrell, D. A., Hultmark, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CecC%2C+a+cecropin+gene+expressed+during+metamorphosis+in+Drosophila+pupae%5BTitle%5D)+AND+%22Eur.+J.+Biochem%22%5BJournal%5D)">CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae.</a> Eur. J. Biochem. 204, 1-5 (1992).</li>
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<li>Cook, P. E., Sinkins, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+profiling+of+Anopheles+gambiae+mosquitoes+for+adult+age+estimation%5BTitle%5D)+AND+%22Insect+Mol.+Bio%22%5BJournal%5D)">Transcriptional profiling of Anopheles gambiae mosquitoes for adult age estimation.</a> Insect Mol. Bio. 19 (6), 745-751 (2010).</li>
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</ol>

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在蚊子引起的非转基因的RNAi目前的方法包括直接注射双链RNA进入成人血腔12,13或RNAi触发包覆的纳米微粒14-17或微藻为基础的dsRNA分子18幼虫取食。针对成蚊，而极有价值的，可以排除大量在早期发育阶段的功能基因。由幼虫喂养发起击倒可通过蛹期期间，由于成年阶段得到不一致的表型，在部分地可变蛋白的持久性的潜力。因此，引入了在蛹开发期间发起的RNAi将提供更加完全地评估期间成人预的发育阶段的基因功能，以及增强的能力时成人阶段来评估基因功能的装置专门针对一个额外的方法。与基于双链RNA注射或表达，基因敲除的持久性基因敲除的方法无法预测。因此，成绩单或蛋白质水平应在感兴趣的发育阶段进行评估感兴趣的基因。虽然我们观察到的延续的降低在5天注射后在SRPN2双链RNA注射的动物SRPN2蛋白水平的因子，如蛋白质周转和半衰期可以为不同的目标不同。关键是要保证，以及，要被用于注射所述dsRNA是很好浓缩并出现在琼脂糖凝胶上完好无损。我们建议实验者重新评估这些因素，如果击倒效果是不够的，并且双链RNA的各自的浓度，可能需要对特定基因靶凭经验测试。 我们描述了在An的蛹期用于RNA干扰的开始的方法。冈比亚发展。此方法依赖于通过dsRNA的显微注射引入直接进入早期蛹的血腔，并且允许通过注射质量的评价使用染料标记的dsRNA。形象化注射质量的能力构成用于确保成功击倒的关键的提高和构成还没有在大多数先前报道基于双链RNA协议侧重于成年阶段被认为是喷射型的基因敲除的一个方面。通过靶向在此发育期间，基因这一关键发育间隔期间可能发挥作用的发作的蛹，或在成年的早期阶段可以在功能上进行评估。此外，这种方法可以使双链RNA递送至细胞中，并建立RNA干扰中是变态期间访问的细胞，但在完全形成成蚊较少访问。 通过哈克等人最近的微阵列分析（2012）确定的560。冈比亚转录物被上调或至少4倍期间不同发育阶段下调，从胚胎到成年。 56的0成绩单标识，一套309被蛹发育期间27上调。这些结果表明，有对差异基因表达许多要求整个蚊发展，包括那些在蛹期所发生的，在此期间，该生物体经历变态的间隔。在许多昆虫物种，包括一种。冈比亚 ，参与诸如发展（ 即蛹表皮和甲壳素结合蛋白）过程的基因27-31和免疫反应（ 即收费受体样蛋白）27,32-34高度在蛹期表达。一旦完全形成成人已经出现，有继续应对环境和生理变化35基因的表达。值得注意的是，早期成年开发期间，有一个增加发育基因（ 例如 ，成人表皮和肌浆蛋白）36，的表达以及其它关键基因（ 即 </em&gt;，精子特异性蛋白和细胞色素P450酶代谢）27,36。 在这个协议中，SRPN2的开发中使用的阳性对照，是一个。冈比亚丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）。 SRPN2起着昆虫黑化，在昆虫21,22广谱先天免疫应答的负调节中起重要作用。在伪肿瘤形成21,22，即很容易被利用光学显微镜的观察到的表型成蚊结果SRPN2的击倒。鉴于这一独特的表型可在现场昆虫轻松得分，我们采用SRPN2初始蛹期的RNAi注射。此外，SRPN2是在所有发育阶段表达37，从而为蛹期的RNAi注入和功能评估好目标在早期的成人。我们表明，我们已经开发的方法能够诱导类似成人黑色素理想跟踪准的<html>邻肿瘤形成的过程中发展的蛹期进行的双链RNA注射的结果。在制定这个协议，我们已经观察到，注射蛹早期发育过程中（ 即幼虫，蛹蜕皮后的第一个24小时）是获得最佳成虫羽化的关键。在出现穷人获得注射后的情况下，我们建议分期幼虫更准确，获得蛹用更少的广泛角质层硬化，并保证早期蛹期注入的实现。此外，在注射过程中最小化对在最佳的出现率角质层结果损坏和增加放大率可帮助确保只有动物的预期区域被刺破。如果针应通过穿刺到腹侧角质层，染料标记的液体的池将在蛹的外部明显或将饱和滤纸。强烈建议丢弃有多个角质层刺穿任何蛹。 jove_content"&gt;随着许多实验室的成人蚊注射剂的性能的广泛的经验，先前确定微注射方法可以适于与简单的协议修改为在蛹RNAi实验中使用。总体而言，该方法的目标是提供研究者的能力展开期间反向遗传分析可以进行，进一步使研究，将支持的新颖矢量控制策略的发展的时间框架。有趣的是，在其它物种，如吸血蝽和草地贪夜蛾的实验中，揭示了该基因沉默效应往往是多当更多的预成年期间开始阶段38,39。在发展的各个阶段，击倒RNAi介导的基因是受到有关速度和基因沉默的持久性，并通过有针对性的基因编码的蛋白质的稳定性考虑，理想的RNAi靶基因往往是那些编码一个protein或RNA具有半衰期短，高周转率11,40。 而转基因的RNAi策略也可使用，以解决在成人预阶段就迅速和RNAi的持久性考虑，转基因技术有很多缺点（ 例如 ，转基因株系的生成所需要的时间，实验时限蚊子交配以产生昆虫与双链RNA监管的表达，和转基因股维护）。相比之下，我们的协议得到了发展蛹期间和源于且变态期间访问，但在成人不易进入细胞类型的启动基因敲除的方便和快捷的方法。使用染料标记的dsRNA混悬液允许注射成功的容易评估和蛹内引入材料的分散。我们对肿瘤形成在蛹注射成人发病数据相比，注射作为成年人的动物，是使用init一致在蛹期iation RNAi介导击倒。使用我们的G3蚊子线和我们的养虫室条件下，我们观察到早10天的成人注射后注入三到五天出苗后成年黑色素伪肿瘤的形成。通过进行早期蛹阶段喷射，我们早在五天后注射（ 即 ，三至四天后出现）观察可见黑色素假性肿瘤的形成。这些数据意味着，这种方法能够在一个以前在深入研究的发展期（ 即蛹发育）的基因敲除的开始。我们的染料标记方法也可能被证明为新幼虫注射协议的发展是有用的，由于角质层的过程中所有的幼虫龄的半透明性质。虽然在本研究中使用的控制需要进展到成年阶段来显示击倒表型，今后的实验，以评估在注射后早期蛹将提供蛹阶段特异性的表型对于这种方法为额外的发育阶段的扩张宝贵的见解，如后期蛹发展。总之，这种方法提供了RNAi介导的基因敲除的蛹期开始了宝贵的RNAi协议，增加可用的矢量昆虫研究社区内利用功能基因组学的工具。

疟疾是一种由蚊子传播的传染病，每年影响数以百万计的个人。世界卫生组织（WHO）报告说，在2013年大约有584000人死于疟疾，78％，其中发生在儿童五年1岁。导致人类疟疾的病原体是疟原虫属内顶复门寄生虫和按蚊雌性蚊子在人类宿主之间的传输。当蚊子采取血粉从谁被感染的个体，然后存款感染寄生虫进入未感染的个体在随后的血粉发生传输。在按蚊属， 冈比亚按蚊是最大的矢量能力的物种，是撒哈拉以南非洲地区1-3最突出的疟疾向量。 目前，由杀虫剂部署蚊虫媒介​​控制继续到bË用来减少人类疟疾负担的主要方法。虽然自上世纪60年代使用杀虫剂已被证明是非常成功的，抗药性的兴起，带动需要新的杀虫剂和替代的媒介控制策略4-7发展。 2010年期间，共有49 63个国家向世卫组织报告的说明杀虫剂抗药性疟疾媒介1的发生。此外，IR映射工具，它采用同行评议的文献，评估Afrotropical地区性数据，报告说，2001年和2012年间有阻力分别为7 46％和27％提高到拟除虫菊酯和滴滴涕。 RNA干扰（RNAi）在90年代初被认定为可能被用来灭活在矮牵牛工厂8,9和真菌孢霉 9,10基因的技术。此后不久，在1998年，RNA干扰最早在秀丽隐杆线虫记录为通过经由注射或进料的方法9,11-引入反义或双链RNA（dsRNA）的的减少在动物模型中的基因表达的一种手段。自从被发现以后，RNA干扰已被使研究人员能够利用反向遗传学通过高选择性转录后基因沉默机制，以迅速调查目的基因的功能角色，彻底改变了功能基因组学的追求。在一些生物如黑腹果蝇 ，使用表达干扰RNA构建体的转基因生物的已经用于基因敲除（KD）广泛的成功。虽然在一个使用转基因的。冈比亚为RNA干扰已被利用，并且可能被证明为大型屏幕是有用的，转基因蚊的菌株的产生是劳动力和时间密集的，一般服用两到三个月从识别的感兴趣的基因的给generati去在合适的转股价12。目前， 一个基因KD的主要方法。冈比亚是通过注射入血淋巴，在成年阶段，dsRNA的特异于给定基因12,13。这个过程通常需要一个月左右，从感兴趣的基因KD的评估基因鉴定去了，证明是比转基因方法12更加迅速。幼虫阶段的RNAi方法一直在一家新近成立的。基于微藻冈比亚和埃及伊蚊通过纳米颗粒喂养14-17或口服给药dsRNA分子18，提供机会在开发的早期阶段进行功能基因组分析。在直接喷射，进料，和纳米颗粒递送方法，双链RNA溶于自主由靶细胞和裂解由酶Dicer的成21-25核苷酸长"短干扰RNA"（siRNA的）19,20。这些siRNA然后incorporated入RNA诱导的沉默复合物（RISC）中，从其中一条链将被丢弃，从而允许所述RNA结合的RISC复合物结合并切割靶mRNA，从而降低其电平，并抑制其翻译19,20。 基本蚊生物学的许多内在特性矢量调制能力，包括主机偏好（ 例如 ，嗅觉，味觉），交配，繁殖和免疫。鉴于这些生物过程的重要性，它很可能是他们的基因或药物水平调节将提供矢量控制的新机遇，包括杀虫剂抗性的规避，以及更广泛的综合方法，以矢量管理提供了新的工具。利用功能基因组学，评估基因基本这些内在的生物学特性的角色将启用新的目标识别和提供新的见解，我们如何才能有效地建立新的，更有效的控制战略中IES。我们描述了在An的蛹期开始的RNAi迅速注入方法的开发和使用。冈比亚 。我们观察到的RNAi触发器的蛹期注射能够产生的表型的观察早期的成年人， 即出现比，如果基因敲除成人发起苗后将观察后越快。此方法使基因拦截蛹发育间隔期间开始并延伸到成人的阶段，使得蛹发展期间启动基因敲除能坚持和影响成年早期血淋巴访问的细胞类型，以及细胞类型是变态期间更血淋巴访问的那比在成年人，例如在以下出现成人附属物发现感觉神经元。

<table><tbody><tr><td>MEGAscript RNAi kit</td><td>Ambion</td><td>AM1626</td><td></td></tr><tr><td>Nanoject II injector</td><td>Drummond</td><td>3-000-204</td><td></td></tr><tr><td>Nanoject II foot switch</td><td>Drummond</td><td>3-000-026</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Drummond</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Glass micropipette puller</td><td>Narishigne</td><td>PB-7</td><td></td></tr><tr><td>Fast Green FCF dye</td><td>Sigma</td><td>F7258</td><td>Can substitute with a non-toxic food dye.</td></tr><tr><td>Plastic transfer pipettes</td><td>Thermo Scientific</td><td>1371150</td><td>&amp;frac14; inch cut from tip to create a wider opening.</td></tr><tr><td>Whatman &amp;ldquo;thin&amp;rdquo; filter paper&amp;nbsp;</td><td>GE Healthcare</td><td>1001-090</td><td>This is 9 cm diameter Grade 1, but can be altered depending on size of injection pad.</td></tr><tr><td>Western blot &amp;ldquo;thick&amp;rdquo; filter paper</td><td>BioRad</td><td>107-3931</td><td>This is the filter paper generally used for Western blots.</td></tr><tr><td>Petri dishes (60 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Paint brush (size 0)</td><td>Michael&amp;rsquo;s Art</td><td>10474940</td><td>Brush size and brand can vary based on availability and user preference.</td></tr><tr><td>Dissecting light microscope</td><td>&amp;nbsp;----&amp;nbsp;</td><td>-----</td><td>This can vary based on availability and user preference.</td></tr></tbody></table>

rnai trigger delivery into anopheles gambiae pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

蛹注射基因KD收率当在早期蛹期进行注射最佳的结果，当角质层晒黑水平低（ 图1A，左和1B）。晒黑增加角质层硬化，一般24小时，导致增加的蛹死亡注射后（图1A，中，右）后。蛹发展的速度可以根据杀虫条件和动物密度24,25变化;因此，最好是在视觉上评估色素沉着。 在注射过程中，毛细管针以约30°的前方后方向（ 图2A）的角度插入到背角质层。一旦针插入和所述dsRNA + 0.01％（重量/体积）的FGD被分配，染料的分布在整个血淋巴是明显的（Figu重新2B）。 对蛹羽化评价注射0.01％（重量/体积）的FGD显示70％出苗的平均速率，相比未注射的对照组（ 图3A）的96.7％出现。值得注意的是，观察到了大量的非存活的蚊子（ 图3B）从蛹壳局部出现。注射的动物表现出出苗时间没有延误（ 图4A）或任性别（ 图4B）偏见的影响。成人生存的其他评估进行了长达10天，出苗后发现在出苗后成人生存（ 图4C）无明显影响。 KD质量验证通过与SRPN2相关击倒21,22作为击倒和阳性对照的黑色素假性肿瘤（重水复疑无路色素组织簇）的表型评估<html>不存在与dsLacZ注射作为阴性对照关联的表型。所出现的成蚊在8天注射后（6-7天出苗后）进行评分。黑色素伪肿瘤（ 图5A和5B）通过的dsSRPN2与93.5％的角质层观察在dsLacZ成蚊（ 图6A）的0％。黑暗组织白化集群对色素斑（ 图5C）的解剖鉴定。伪肿瘤对成人角质层早3天苗后均可见和也存在于dsSRPN2血淋巴和肠组织中的一个子集（未示出数据）。在5后喷射（成年早期阶段），显著在dsSRPN2下降SRPN2水平，但观察到未dsLacZ或非注入血淋巴分离蛋白（ 图6B）。 ftp_upload / 53738 / 53738fig1.jpg"/&gt; 图1:蛹的dsRNA注射发育分期的dsRNA导致最佳存活和进展到成年期的早期蛹注射。角质层的色素沉着水平低（A，左，和B）可以在第一0-24小时以下化蛹内被观察到。生存贫困人口的蛹表皮注射前（A，中，右）的结果适中晒黑。 <img alt="图2" src="/files/ftp_upload/53738/53738fig2.jpg" /> 图2:注射位置和染料标记的双链RNA（A）的毛细管针注射染料标记的dsRNA进入背角质层的分布在约30°的角度，在从前到后的方向。 （B）的该染料明显地分布在蛹血淋巴。的dsRNA注射体积138 NL的，标有0.01％FGD（重量/体积）。 <img alt="图3" src="/files/ftp_upload/53738/53738fig3.jpg" /> 图3:后喷射成虫羽化 （A）的蛹的70％与0.01％的FGD注射（重量/体积）在成功出现（N = 60），相对于未注射的对照（n = 60）的96.7％。进行三次生物学重复。观察到大量非存留蚊子（B）从蛹壳部分出现。误差棒代表平均值（SEM）的标准误差。 <img alt="图4" src="/files/ftp_upload/53738/53738fig4.jpg" /> 图4:出苗速度，性别评估和成人存活 （A）中观察到可比出现倍以下蛹注射含0.01％的FGD（24小时= 80％和48小时= 20％），相比于未注射蛹（24。 HR = 83％和48小时= 17％）。 （B </str翁&gt;）观察到0.01％的FGD注射蛹（女性= 48％和男性= 52％）和未注射的蛹（女性= 52％和男性= 48％），大约等于雌雄成虫羽化。 （C）生存分析结果显示，注射0.01％FGD不影响成人生存，评估多达10天出苗后。结果从含0.01％的FGD三个独立实验注射（60例）和未注射的（60）蛹（男性和女性的相等数目）表示的数据。误差棒代表平均值（SEM）的标准误差。 <img alt="图5" src="/files/ftp_upload/53738/53738fig5.jpg" /> 图5:伪肿瘤阳性对照表型反映成功的击倒上观察到的（A）的腹部和dsSRPN2的（B）的胸椎角质层-injected伪肿瘤，但不dsLacZ在第8天注射后-injected成人蚊子。（C）高倍（400X）角质层和色素斑块的解剖成像（A）（B）揭示黑暗变暗细胞群。 <img alt="图6" src="/files/ftp_upload/53738/53738fig6.jpg" /> 图6:假性肿瘤的形成量化，降低SRPN2蛋白水平 （A），蛹期注射导致假性肿瘤形成的dsSRPN2成年人93.5％（N = 21）相比，dsLacZ对照为0％（N = 19）。 。结果得到的第8天后注入。 （B）蛋白质印迹（左）的节目在dsSRPN2降低SRPN2水平，但不dsLacZ或非注入血淋巴分离蛋白（第5天注射后）。结果基于三个独立的实验。使用的抗SRPN2 21和抗SRPN3 26抗体稀释度为1:分别2,000:1,000和1。山羊抗 - ラ5000:BBIT的IgG-HRP（产品SC-2004，Santa Cruz公司，达拉斯德克萨斯州）1使用。所有蛋白水平进行定量（右）通过条带强度（ImageJ的软件，美国国立卫生研究院，贝塞斯达，MD）归一化到SRPN3，和统计由非配对t检验进行比较。 P &lt;0.05 *，P≥0.05:NS（不显著）。误差棒代表平均值（SEM）的标准误差。

Watch this Scientific Journal Video about RNAi Trigger Delivery into Anopheles gambiae Pupae at JoVE.com

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

rnai-trigger-delivery-into-anopheles-gambiae-pupae

Nanyang Technological University

Research

JoVE Journal

Biology

RNAi Trigger Delivery into Anopheles gambiae Pupae

9.1K 次观看.  Boston College. RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

视频: RNAi Trigger Delivery into Anopheles gambiae Pupae

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

女答：埃及伊蚊蚊的dsRNA注入

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

生物学

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

环境科学

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

冈 比亚按蚊 胚胎的显微注射方法

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations

Source: Carballar-Lejaraz&#250;, R. et al, <a href="https://www.jove.com/v/62591">Microinjection Method for Anopheles gambiae Embryos.</a> J. Vis. Exp. (2021).
In this video, we demonstrate the delivery of gene drive construct into the embryo of Anopheles gambiae mosquito through embryo microinjection technique. This procedure allows for integration of&#160; delivered DNA construct, through homologous recombination, into the germline cells to generate transformed mosquito population.

蚊子胚胎显微注射:一种将外源 DNA 递送到冈比亚按蚊胚胎卵黄中以产生种系突变的技术

Source: Carballar-Lejaraz&#250;, R. et al, Microinjection Method for Anopheles gambiae Embryos. J. Vis. Exp. (2021).
In this video, we demonstrate the ...

RNAi的触发传送到

摘要

探索更多视频

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

女答：埃及伊蚊蚊的dsRNA注入

成蚊（冈比亚按蚊的RNAi实验议定书）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

冈比亚按蚊胚胎的显微注射方法

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

蚊子胚胎显微注射:一种将外源 DNA 递送到冈比亚按蚊胚胎卵黄中以产生种系突变的技术

RNAi的触发传送到

摘要

探索更多视频

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

女答：埃及伊蚊蚊的dsRNA注入

成蚊（冈比亚按蚊的RNAi实验议定书）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

冈 比亚按蚊 胚胎的显微注射方法

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

蚊子胚胎显微注射:一种将外源 DNA 递送到冈比亚按蚊胚胎卵黄中以产生种系突变的技术

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

冈比亚按蚊胚胎的显微注射方法

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药