我们感谢中心的生物信息学和功能基因组学（CBFG）在迈阿密大学为技术依托。这项工作是由美国迈阿密大学开办补助（YT），以及美国国家科学基金会（:IOS 0950964 YT）的支持。

1，T。盗股票和培养<ol><li>决定一个T。盗菌株用于该试验。 注:有几个实验室建立的T。盗菌株是可用的。 嘎-1（佐治亚-1）为Ga-2亲本菌株用于基因组测序3这一点。由于Ga的1股大部分的DNA多态性（如单核苷酸多态性，单核苷酸多态性）与镓-2，并且比Ga的2健康由于较少近亲繁殖，这是理想的RNAi实验。PU-11是另一种方便的菌株用作为它的独特的增强型黄色荧光蛋白（EYFP）表达在未来翅原基使我们能够区分来自其它龄期的最后的幼虫龄期（见的克拉克-Hachtel 等人 2013 12 图S4）。它记录其甲虫菌株在实验中使用是很重要的，因为遗传背景出现显著AFF等了RNAi的表型13。 </li><li>准备T。通过加入预先过筛的啤酒酵母的5％（按重量计），以有机全麦面粉（混合后，储存在-20℃下培养面粉） 盗培养面粉。等分的培养面粉（在室温下）为6盎司塑料果蝇库存瓶（约40克/瓶）。 </li><li>文化T。拟谷盗 ，在30℃用70％的湿度。使用湿度控制孵化器如果可能的话。添加30-40成年人每培养瓶（男女比例1:1），开始培养。 注意:虽然模具是很少的问题，在30℃下，较低的温度（如25℃），用70％的湿度会导致霉菌生长在培养面粉。降低湿度，如果发生这种情况。 </li><li>转大人到一个新的培养瓶每两周传代培养（参见如何隔离成年人从文化面的步骤5.2-5.5）。 注意:这需要大约三个星期，以获得末龄幼虫。 Alternat结构延续，T。盗培养物可以保持在较低的温度下（20-25℃），尽管更长的培养时间（发育时间在25℃时是大约两倍于30℃下）。 </li></ol> 2，准备解决方案和仪器幼虫双链RNA注射液<ol><li>合成的dsRNA分子的同源性通过体外转录的靶基因。储存在-80℃。看到菲利普和泰2011 11进行详细的分子生物学方法。另请参阅讨论了一些需要加以考虑设计的双链RNA分子，当参数。 </li><li>通过混合8.5毫升的1M的Na 2 HPO 4用1.5ml的1M的NaH 2 PO 4的制作将10ml 0.1M磷酸钠缓冲液（pH 7.6，在25℃）。检查用pH计的pH值，并相应地调整。 </li><li>使加入1ml 10×喷射缓冲器通过将10微升的0.1M磷酸钠缓冲液（pH 7.6，在25℃），100 _6;升0.5M的氯化钾，加入100μl的绿色食用色素，和790微升双蒸水（DDH 2 O）。稀释10倍注射缓冲液，使2个注射缓冲液（200微升10X注入缓冲液+ 800微升DDH 2 O）。同时存储10倍和2倍注射液缓冲在4°C，直到需要的。 </li><li>准备粘载玻片上。 <ol><li>支付与重新定位的胶水幼虫注射一种形式的整个载玻片。对于成人或蛹注射，使两个薄带的胶水沿着滑动的长边。 </li><li>使胶水干燥数天，这保证了胶保持足够粘性附着甲虫在滑动的同时，也保持足够的柔性，以方便注射后除去它们。 注:如果胶水停留太粘，用手指轻点上胶，这将减少粘性。每个滑动可以容纳多达40个或50个甲虫，并且可以重复使用，直到它不能可靠地保持动物。 ALTERNatively，双面胶带也可以使用，虽然在磁带有时无粘性足以容纳幼虫。 </li></ol></li><li>做一个醚化篮。 <ol><li>除去从10ml的一次性注射器的柱塞，并切断所述注射器中的一半左右的6毫升线。 </li><li>弃去底部的注射器的一半。简要地加热用气体燃烧器将注射器的顶端部分的切割表面，并迅速推向熔化塑料到一张尼龙网的粘合网眼到注射器中。修剪掉多余的网状一旦塑料变硬。 </li></ol></li><li>准备乙醚瓶。加入30ml乙醚成窄口100或250 ml玻璃瓶。加入几片纸巾，以提高醚的蒸发。 注:乙醚提出了火灾隐患，也是有害的。在通风橱总是处理醚。紧密而无法合上盖子使用。 注:冰可以作为一个更安全的替代（如在课堂环境中），虽然sedation是不那么有效。 </li></ol> 3，制备幼虫喷射装置<ol><li>发布XY机械平台上解剖显微镜。 注:XY机械阶段，可从主要的显微镜公司。备选地，廉价显微镜阶段也可用于大多数解剖显微镜具有一些修改（见材料表为例子）。 </li><li>广场附近的XY机械舞台机械针操纵。 </li><li>组装的注射器。使用四通活栓（带Luer连接）到30ml的一次性注射器连接到玻璃针保持架，使操纵注射器的，而不会影响在注射针的压力（见菲利普和泰2011 11为详细注射器组装）。 </li></ol> 4，牵引注射针头<ol><li>由拉硼硅玻璃针（0.5毫米，长15厘米B100-50-15，外径:1毫米，ID）针拔。 注:对于萨特的P-87或P-97，使用设置"热火= 70，拉= 45，威赛= 75，时间= 90"，或按照移液器食谱第2章:贴壁细胞，C。线虫 ， 果蝇 ， 斑马鱼及-推荐程序。最佳设置取决于针拉马的模型。设定一个通用的果蝇注射针是一个很好的起点。 </li><li>商店拉针在塑料外壳（ 如塑料CD盒），并带有可拆卸安装腻子固定。 </li></ol> 5，分离与筛选幼虫<ol><li>放置一个＃25筛上筛接收器。 </li><li>将培养瓶（甲虫和培养面粉）在筛上的内容，并立即过筛的内容。不要留在筛上的内容而不进行筛选，因为幼虫会迅速尝试挖洞通过筛板和卡住。积极筛选的内容，让面粉下跌通过和离开旧的幼虫（一般为6 次和第 7 次龄幼虫），蛹和成虫（连同其抛弃的角质层，蜕皮）后在筛上的顶端。 </li><li>传送保留在筛（甲虫和蜕皮）到种子盘的材料。请敲击锅，以防止甲虫逃逸。 </li><li>取出蜕皮和轻轻拂动种子盘剩余的甲虫表面的面粉颗粒。 </li><li>点击种子潘移动内容到锅底。然后，离开盘未开发的几秒钟。等待大人，这比其他级以上移动，以从堆出来。通过使用艺术画笔（ 如 1厘米宽）删除成年人。 </li><li>独立蛹轻轻敲锅种子，同时保持锅内的嘴角微微向下，因为蛹倾向于更快地推出对嘴。用画笔去除蛹，在种子盘只剩下幼虫。 </li><li> PLACE在一个干净的培养皿中分离出幼虫。选择幼虫的适当阶段注射，并将其放置在一个单独的培养皿中。 注:早期末龄幼虫（1-2天的最后幼虫蜕皮后）当分析对成人的形态，以及对变态的RNAi效应常常是合适的。据，但是，有时有必要在倒数第二执行RNA干扰（或更早）阶段来评估早期基因功能（ 例如， 图3E-3H，也可参见克拉克-Hachtel 等人 2013 12）。使用蛹作为大小参照来确定幼虫的龄期。末龄幼虫略长于蛹。备选地，PU-11可用于识别末龄幼虫以及确定末龄幼虫的发展（克拉克-Hachtel 图S4 等人 2013 12）的时间过程。 </li><li>请将选定的幼虫在培养皿中，直到醚化。将剩下的幼虫和甲虫的其他阶段（如蛹和成虫），回到一个培养瓶用面粉，并返回到孵化器。 </li></ol> 6，准备注射针头的<ol><li>将以前拉玻璃针使用上既黏粘或双面胶带显微镜载玻片。使用镊子，测试针拔出，并同时期待通过解剖显微镜才能看到顶端弯曲。稍微抢区朝那里的针弯曲的镊子和扭曲打破了尖端的一面。 </li><li>保持良好的针头和丢弃等。考虑好针为具有开口，既不太大也不能太小（约0.05mm的直径。 图1A和1B），并成角度以使幼虫角质层容易（ 图1B和图2A-2B）的渗透。考虑一个坏的针是（i）过小，使得双链RNA溶液吸收进针diffi崇拜（步骤7）（ 图1D和2D），（ⅱ）过大时，在注射时引起幼虫损伤和可能的致死率（ 图1C和图2F），（ⅲ）钝，使得角质层困难和增加损伤的渗透。 </li><li>针插入持针器。 <ol><li>首先，拧下针保持架的前端和放置针的后端插入支架小费。然后，将橡胶垫圈的保持器前端下（至少为1厘米的玻璃针的后端）。 </li><li>插入针的背面插入支架，用橡胶密封垫完全插入针保持器的开口。螺钉尖端放回支架。 </li></ol></li><li>将组装的缝合针夹持器上的针操纵器。保持针保持接近水平的。通过MICR看时调整操纵器的位置，因此，针的末端是位于视图的中心oscope需要。 </li></ol> 7，前期投资的双链RNA解进针<ol><li>通过调整与DDH 2 O的浓度和混合用的2倍注射缓冲液（2.3步）等体积的双链RNA溶液制备的双链RNA注射液之前，为了注入。保持在冰上的混合溶液中。看到有关的dsRNA浓度的讨论。 </li><li>砍20微升一次性吸头的尖端。移取10微升的溶液进入修整枪头。从移液管，同时保持流体在前端通过设置背压小心取出枪头。备选地，小心地取出前端，然后通过使用手指提供压力将溶液到尖端的末端。 </li><li>将移液管尖端与使用粘性粘性朝向所述针的前端开口部的显微镜载物台的dsRNA的溶液。 </li><li>翻翻显微镜，缓慢移动装尖向针，直到针尖针只是里面加载的枪头和进入溶液。 </li><li>同时期待通过显微镜，轻轻一拉回到了注射器的柱塞慢慢地将双链RNA解进针。 注意:不要超载针。作为dsRNA的溶液的末端接近针的前端，慢的拉动速度和停止加载的针的前端之前的溶液将达到空气。如果空气到达前端时，溶液会迅速被吸入针座，从而导致严重的污染问题。针座的详细清洗程序在菲利普和泰2011 11进行说明。 </li><li>通过打开旋塞除去从注射针筒和针固定器中的压力。然后，从该针的前端移动的移液管尖的路程。从舞台升起针，以防止意外损坏针，同时将幼虫在舞台上。卸下舞台上的走光枪头。 </li></ol&gt; 8双链RNA注射液<ol><li>将幼虫放入乙醚麻醉篮筐。使用不到10个幼虫，如果学习的技术。使用30-40幼虫在一个回合一次舒服的方法。 </li><li>把篮子幼虫在乙醚瓶，并盖上盖子。 注:乙醚提出了火灾隐患，也是有害的。执行在通风橱这一步。 </li><li>醚化幼虫约3分钟。检查幼虫的运动3分钟后。将他们带回了一瓶乙醚另一个30秒，如果他们还在移动。不要醚化时间超过5分钟，因为这可能会增加杀伤力。 注:根据温度和湿度的最佳醚化时间可能会有所不同。 </li><li>从篮子传送醚化幼虫的玻璃粘滑动的不粘边。使用镊子和同时期待通过显微镜，拿起每一个幼虫逐个把它们拖到幻灯片。把它们放横向和轻轻拍打了他们的身体˚FROM头尾用钳，稍微伸展它们，当您去，以确保它们是固定在载玻片上。 </li><li>将粘带滑幼虫在显微镜阶段。 </li><li>将双链RNA装针轻轻放入幼虫的背侧以避免损坏中枢神经系统。此外，还要避免背中线，随着幼虫心脏就设在这里。在这和随后的步骤，请务必使用阶段的幼虫移动（而不是使用针操纵移动针）的针。 注:具体注射部位没有关系的RNAi技术在T。盗似乎是全身性的。然而，它通常是最容易注入到胸部或腹部段的背侧。 </li><li>将针插入幼虫后，拔出针头稍回，让空间的双链RNA进入幼虫体腔。 </li><li>轻轻一推就注射针筒，直至幼虫变成绿色（或注射缓冲区的颜色），并期待Štretched饱满。 注:如果学习的技术中，尝试注入尽可能确定的解决方案，它可以被注射到幼虫无显著杀伤力的量。它通常为约0.5-0.7微升。 </li><li>取下幼虫针。当拆下针，稍微拉回来的注射器，以避免损失的dsRNA（也注意不要拉在空气中）。 </li><li>注幻灯片上所有的幼虫。请务必删除幼虫未成功注入。完成注射幼虫前醒来（约10分钟）。 </li><li>从注射显微镜移除与注射的幼虫滑动并离开滑块5分钟以上，直到从乙醚麻醉所有幼虫回收。 </li><li>用镊子和解剖显微镜下，轻轻地从头上幼虫提升到尾部，从粘性滑动释放他们。放置在一个干净的培养皿中新发布的幼虫。留在培养皿中的幼虫10-15分钟，以允许注射瓦特ound凝结。 </li><li>将幼虫放置到一个新的瓶用干净的面粉和标签适当地包括菌株名，dsRNA的类型注入浓度的dsRNA，幼虫注射编号和日期。 </li><li>培养在30℃下注入的幼虫，用70％的湿度。观察幼虫定期的RNAi表型。 注:在7天末龄幼虫化蛹。然后，蛹将E关闭到7天后成虫（如果在定时没有异常引起的RNAi）。 </li></ol> 9，确认的击倒和表型分析<ol><li>考虑的RNAi的效率评估由几种不同的分子生物学技术，如定量反转录-PCR（QRT-PCR）和免疫印迹击倒。见Miller 等人2012年7菲利普和泰2011 11进行详细的定量PCR和Western Blot协议，分别为。 </li><li>分析相关的RNAi表型。 注:相关的RNAi表型可以在几个不同的阶段，不同的培养条件下观察到的，这取决于被靶向的基因。 RNA干扰会影响甲虫形态，变态，生理和行为。通过RNA干扰的频率表型的存在进行检查和在什么条件下的甲虫饲养应针对具体问题正在调查中。 </li></ol>

<ol>
	<li>Denell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Tribolium+as+a+genetic+model+system+and+its+early+contributions+to+evo-devo.">Establishment of Tribolium as a genetic model system and its early contributions to evo-devo.</a> Genetics. 180, 1779-1786 (2008).</li><li>Klingler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tribolium.">Tribolium.</a> Curr Biol. 14, R639-R640 (2004).</li><li>Richards, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+the+model+beetle+and+pest+Tribolium+castaneum.">The genome of the model beetle and pest Tribolium castaneum.</a> Nature. 452, 949-955 (2008).</li><li>Bucher, G., Scholten, J., Klingler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+RNAi+in+Tribolium+(Coleoptera).">Parental RNAi in Tribolium (Coleoptera).</a> Curr Biol. 12, 85-86 (2002).</li><li>Tomoyasu, Y., Denell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+RNAi+in+Tribolium+(Coleoptera)+for+analyzing+adult+development.">Larval RNAi in Tribolium (Coleoptera) for analyzing adult development.</a> Dev Genes Evol. 214, 575-578 (2004).</li><li>Tomoyasu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+systemic+RNA+interference+in+insects+a+genome-wide+survey+for+RNAi+genes+in+Tribolium.">Exploring systemic RNA interference in insects a genome-wide survey for RNAi genes in Tribolium.</a> Genome biology. 9, R10 (2008).</li><li>Miller, S. C., Miyata, K., Brown, S. J., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+systemic+RNA+interference+in+the+red+flour+beetle+Tribolium+castaneum+parameters+affecting+the+efficiency+of+RNAi.">Dissecting systemic RNA interference in the red flour beetle Tribolium castaneum parameters affecting the efficiency of RNAi.</a> PLoS ONE. 7, e47431 (2012).</li><li>Tomoyasu, Y., Arakane, Y., Kramer, K. J., Denell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+co-options+of+exoskeleton+formation+during+wing-to-elytron+evolution+in+beetles.">Repeated co-options of exoskeleton formation during wing-to-elytron evolution in beetles.</a> Curr Biol. 19, 2057-2065 (2009).</li><li>Miller, S. C., Brown, S. J., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+RNAi+in+Drosophila.">Larval RNAi in Drosophila.</a> Dev Genes Evol. 218, 505-510 (2008).</li><li>Brown, S. J., Mahaffey, J. P., Lorenzen, M. D., Denell, R. E., Mahaffey, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNAi+to+investigate+orthologous+homeotic+gene+function+during+development+of+distantly+related+insects.">Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects.</a> Evolutio., &amp; development. 1, 11-15 (1999).</li><li>Philip, B. N., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+knockdown+analysis+by+double-stranded+RNA+injection.">Gene knockdown analysis by double-stranded RNA injection.</a> Methods Mol Biol. 772, 471-497 (2011).</li><li>Clark-Hachtel, C. M., Linz, D. M., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+insect+wing+origin+provided+by+functional+analysis+of+vestigial+in+the+red+flour+beetle.">Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle.</a> Tribolium castaneum, Proceedings of the National Academy of Sciences of the United States of America. 110, 16951-16956 (2013).</li><li>Kitzmann, P., Schwirz, J., Schmitt-Engel, C., Bucher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+phenotypes+are+influenced+by+the+genetic+background+of+the+injected+strain.">RNAi phenotypes are influenced by the genetic background of the injected strain.</a> BMC genomics. 14, 5 (2013).</li><li>Parrish, S., Fleenor, J., Xu, S., Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+a+dsRNA+trigger+differential+requirement+for+the+two+trigger+strands+in+RNA+interference.">Functional anatomy of a dsRNA trigger differential requirement for the two trigger strands in RNA interference.</a> Molecular cell. 6, 1077-1087 (2000).</li><li>Winston, W. M., Molodowitch, C., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Systemic RNAi in C elegans requires the putative transmembrane protein SID-1. 295, 2456-2459 (2002).</li><li>Tomoyasu, Y., Wheeler, S. R., Denell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabithorax+is+required+for+membranous+wing+identity+in+the+beetle+Tribolium+castaneum.">Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum.</a> Nature. 433, 643-647 (2005).</li><li>Trauner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+insertional+mutagenesis+of+a+coleopteran+stored+grain+pest,+the+red+flour+beetle+Tribolium+castaneum,+identifies+embryonic+lethal+mutations+and+enhancer+traps.">Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps.</a> BMC Biol. 7, 73 (2009).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Drosophila+retinal+determination+gene+network+in+Tribolium+(II)+The+Pax6+genes+eyeless+and+twin+of+eyeless.">Probing the Drosophila retinal determination gene network in Tribolium (II) The Pax6 genes eyeless and twin of eyeless.</a> Developmental biology. 333, 215-227 (2009).</li><li>Horn, T., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-RNAi+a+web+application+for+the+multi-species+design+of+RNAi+reagents--2010+update.">E-RNAi a web application for the multi-species design of RNAi reagents--2010 update.</a> Nucleic acids research. 38, 332-339 (2010).</li></ol>

作者什么都没有透露。

<html>也有一些需要加以考虑，以保证RNAi技术的成功的重要问题，包括dsRNA分子的不同的dsRNA分子之间的长度和浓度，竞争（尝试多次当击倒），和脱靶效应的可能性（OTE）。 长的dsRNA dsRNA分子的长度会影响全身的RNAi反应的效率，具有较长的dsRNA是更有效的触发RNAi的7,14,15（虽然dsRNA的较长的限制是目前未知）。 dsRNA的长度需要大于50碱基长以诱导有效的RNAi中的T。盗 7。 150个基点和500个基点的双链​​RNA似乎是理想的RNAi实验。尽管较长的dsRNA分子也可以使用，它们将必须OTE的机会增加和基因的克隆步骤将变得越来越困难。 双链RNA浓度ve_content"&gt;不同程度的基因敲低可以根据dsRNA的浓度来实现。1微克/微升似乎是一种合理的起始浓度，这往往会产生近零的表型（可根据感兴趣的基因（S）上的变化）。RNAi技术可以用较高浓度（ 如 7-8微克/微升）进行，以获得更强的RNAi表型的RNAi与dsRNA的连续稀释，有时是有益的，产生了一系列亚效等位基因的表型（泰的补充资料等 2009年8月，和BORRAS -卡斯特未发表的数据人）。 RNA干扰比赛多基因敲除可在T中完成盗通过同时注入多种不同的双链RNA分子。然而，还已知的是具有若干不同的dsRNA分子在生物体中的存在经常导致双链RNA之间竞争的访问的RNAi组分<s达&gt; 7,14。尝试多种基因敲除时，为避免1的dsRNA进行竞争的其他（使用相同长度和相同的浓度为所有的dsRNA是很重要的，虽然，可能需要所述dsRNA长度和浓度的进一步调整时的表达水平之间有很大的差别靶基因）。我们，以及其他人，都成功地进行双重和三重击倒（ 例如 ，泰等人 ，2005年16日 ，泰等人，2009年17，杨等人，2009年18）。虽然可行，四倍的RNAi（或更多）可能是具有挑战性的，因为它可能会导致所有四个靶基因显著还原的RNAi的效率。 离目标 OTE是一种内在的关注基于RNAi的方法。以最小化OTE单程是设计的dsRNA时，以确定与靶基因的区域共享与其他基因相似的序列，并避免这些区域。对T.简单的BLAST分析盗预测基因组可以找出这样的地区。一些在线工具也使潜在的OTE（ 例如，E-19的RNAi）的评价。进行RNA干扰的目标基因的两个不重叠的区域是一个简单而有效的方法来消除这种观察的表型是由OTE引起的可能性。 OTE的可能性最小化，如果RNAi的两个非重叠区产生相同的表型（除非两个非重叠区域有着相似的序列）。 评价基因通过除表型分析等拦截往往临界有效存在的RNAi相关的数据。评估的基因敲除两个主要方面是定量RT-PCR和Western blot分析。 QRT-PCR是一种方便的方式来测量靶mRNA的水平，并且在许多的RNAi相关的研究中，包括那些在T.已使用拟谷盗 （参见Miller 等人 2012 7为例）。 Howev呃，一定要小心，因为我们最近看到一些案件中，靶mRNA水平上调RNA干扰（虽然蛋白质产物被下调）（BORRAS-卡斯特未发表的数据）。现在的时间是未知的，如果这种RNA干扰诱导表达上调可广泛或独特的某些基因。 Western blot分析是另一种方式，以确认基因敲除。这种方法是非常可靠，因为它测量的最终蛋白质产物的量。对靶基因的蛋白产物的特异性抗体的要求是一个缺点，以这种方式。利用多个独立的测量除表型分析，将增加通过RNAi为基础的分析所获得的表型数据的信心。 自T.概念拟谷盗 ，RNAi技术已主要被用来研究基因功能的发展和形成图案。这些T。赤拟谷盗发育研究已在CHARAC非常成功terizing基因的进化上保守和分歧的功能（在Denell 2008年1 Klingler 2004 2审查）。在T。然而，基于RNAi的研究盗不限于发育生物学。例如，RNAi技术可以用来研究基因功能的一系列生理和行为反应，包括紧张性，掠夺，侵略，择偶，活动规律和防御机制。 应用RNA干扰到这些环境中的一个难点是多效性影响的可能性。通常情况下，目的基因会产生多种遍及吨角色赤拟谷盗的生命周期，从而使除去的基因没有意外的表型效应的困难。然而，要在不同阶段方便地进行RNA干扰的能力，往往是一个有效的策略，以避免这些多效性。例如，在成人中，代替幼虫或蛹进行RNA干扰可能使我们能够规避UNIN早期发育过程中往往造成基因敲除的杀伤力。在T的RNA干扰应答的灵活性盗从而使得这款机型最吸引人的适应RNA干扰基因功能的生理和行为反应的实验。 该T。盗系统也非常适用于教学实验室使用。T。盗可在常温（25℃），无需频繁传代面粉/酵母液很容易培养，并且在T中的RNAi技术赤拟谷盗是很简单的，以适应实验室年轻，学习科学家。作为RNAi的是变中的各种生物领域的一个重要的技术，这是至关重要的学生接触到这种技术。在T.幼虫RNAi技术的直进性盗也鼓励更多的学生参与研究，使T。盗为导向的遗传系统，教室的主要候选人。&lt;/ P&gt;

An erratum was issued for <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/52059">Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum</a>. There was a typo in the settings for Sutter P-87 in step 4.1.

An erratum was issued for <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/52059">Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum</a>.

Erratum: Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

红色面粉甲虫， 赤拟谷盗 ，是获得在生物学各领域的普及，部分是由于容易进行RNA干扰（RNAi）1-3因。基于RNAi的基因敲除技术允许科学家进行，而无需使用复杂的遗传方式丢失的功能分析。T。盗显示出强大的全身性RNAi的反应，使得有可能通过简单的注射的双链RNA（dsRNA）的执行的RNAi的任何阶段进甲虫的体腔4-6。多个基因同时敲也是T.可行盗注射两种或更多种不同的dsRNA分子在同一时间7,8。此外，可以通过减少注射的dsRNA 8的浓度来产生一系列亚效等位基因表型。这些特性使基于RNAi的反向遗传技术有吸引力的替代品，以吨传统的正向遗传学拟谷盗 。因为几乎所有T。赤拟谷盗的组织很容易受到细胞外dsRNA分子9，这种技术可以让研究人员研究各种在不同的环境下组织的。此外，尽管本报告侧重于在T中进行RNA干扰拟谷盗 ，此处描述的许多程序也适用于其它的昆虫。因此，该协议是有用的那些谁愿意履行其在T的利益背景下亏损的功能分析拟谷盗 ，以及为有意申请的基于RNAi技术等昆虫的研究人员。 双链RNA注射到幼虫可在各种甲虫的生命阶段，包括幼虫，蛹功能分析和成虫期4,5,10。之前我们已经报道了整体幼虫的RNAi协议，包括分子生物学方法11。在本报告中，我们重点介绍了双链RNA注射的程序，这是最好的视觉助手解释。我们提供详细的一步一步的注入程序，以及好的和坏的注射例子。这种视觉协议补充了我们之前的协议，合并的时候，他们提供的幼虫RNA干扰过程中T的更全面的观点拟谷盗 。此外，我们将讨论的dsRNA分子可能影响RNAi技术，应用的基于RNAi的测定法对于生理研究的成功，以及在教学实验室幼虫的RNAi协议的适用性参数。

<table><tbody><tr><td>Organic whole wheat flour&amp;nbsp;</td><td>Heartland Mill Inc. Kansas</td><td>G1</td><td></td></tr><tr><td>Brewer&amp;rsquo;s yeast</td><td>MP Biomedicals</td><td>2903312</td><td>Sift yeast with #35 stainless sieve before use. Wear protective mask and cover the sieve with plastic wrap, as the sifted yeast is a fine particle and respiratory hazardus.</td></tr><tr><td>6 oz plastic &lt;em&gt;Drosophila&lt;/em&gt; stock bottles</td><td>Fisher Scientific</td><td>11-888</td><td></td></tr><tr><td>Sieve</td><td>Fisher Scientific&amp;nbsp;</td><td>04-881N</td><td>8 in. dia. x 2 in.D. Use #25 (Nominal opening 710 &amp;micro;m) for larvae, pupae, and adults.&amp;nbsp;</td></tr><tr><td>Sieve</td><td>Fisher Scientific&amp;nbsp;</td><td>04 881 10P</td><td>8 in. diameter #35 (Nominal seive opening: 600 &amp;micro;m)&amp;nbsp; Stainless Steel Sieves, 8 in. dia. x 2 in.D. This sieve is ideal to remove clumps from yeast powder.</td></tr><tr><td>Sieve receiver</td><td>Fisher Scientific&amp;nbsp;</td><td>04-866B</td><td>8 in. diameter</td></tr><tr><td>1.5 Quart spouted sample pan&amp;nbsp;</td><td>Seedburo Equipment Co.</td><td>Model 33</td><td>collection pan</td></tr><tr><td>Incubator</td><td>BioCold Environmental Inc</td><td>BC26-IN</td><td>Keep it 30 &amp;deg;C with 70% humidity.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>S374500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S397-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific&amp;nbsp;</td><td>P217-500</td><td></td></tr><tr><td>Food dye&amp;nbsp;</td><td>Kroger</td><td></td><td>green, blue, or red preferable</td></tr><tr><td>Microscope glass slide&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>22-038-103</td><td></td></tr><tr><td>Tack-It Over&amp;amp;Over</td><td>Aleene&#039;s</td><td></td><td>Repositionable glue for sticky slides. Double sided tape can be used as an alternative, however, we found that the adhesiveness varies.</td></tr><tr><td>Plastic CD case</td><td>Amazon</td><td></td><td></td></tr><tr><td>Boroslilicate glass capillary</td><td>Sutter Instrument</td><td>BF100-50-15</td><td>O.D. 1 mm, I.D. 0.5 mm, 15 cm. Without filament</td></tr><tr><td>Needle puller</td><td>Sutter Instrument</td><td>P-87 or P-97&amp;nbsp;</td><td></td></tr><tr><td>Removable mounting putty&amp;nbsp;</td><td>Loctite Fun-Tak</td><td></td><td></td></tr><tr><td>Compressed gas duster</td><td>OfficeMax</td><td>OM96091</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tool</td><td>11231-30</td><td>Dumoxel #3 (to manipulate beetles)</td></tr><tr><td>Forceps</td><td>Fine Science Tool</td><td>11252-20</td><td>INOX #5 (to break needle tips)</td></tr><tr><td>Ethyl ether, anhydrous&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>E138-500</td><td></td></tr><tr><td>Nylon mesh</td><td>Flystuff/Genessee Scientific</td><td>57-102</td><td>120 &amp;micro;m pore size/49% open area</td></tr><tr><td>Media bottle, 100 ml</td><td>VWR</td><td>89000-926</td><td></td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td></td><td>SteREO Discovery V12. Injection microscope.</td></tr><tr><td>Stereomicroscope</td><td>Fisher/Zeiss</td><td>12-070-513&amp;nbsp;</td><td>Stemi2000. Use to break the needle and place larvae onto the sticky slide.</td></tr><tr><td>X-Y mechanical stage&amp;nbsp;</td><td>Zeiss</td><td>4354600000000000</td><td></td></tr><tr><td>X-Y mechanical stage&amp;nbsp;</td><td>Microscopenet.com</td><td>A512</td><td>Inexpensive alternative</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>M-152</td><td></td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1&amp;nbsp;</td><td></td></tr><tr><td>Glass capillary holder</td><td>Narishige</td><td>IM-H1&amp;nbsp;</td><td></td></tr><tr><td>30 ml Disposable syringe</td><td>BD syringe</td><td>309650</td><td>BD Luer-Lok Tip</td></tr><tr><td>Four-way stopcock&amp;nbsp;</td><td>Cole-Parmer Instrument Co.</td><td>EW-30600-03</td><td>Stopcocks with Luer connections; 4-way; male slip</td></tr><tr><td>Art paint brush</td><td>Amazon</td><td>Art Advantage Oil and Acrylic Brush Set, 24-Piece</td><td>Any general paint brush will work.</td></tr></tbody></table>

larval rna interference in the red flour beetle, tribolium castaneum

对赤拟谷盗， 赤拟谷盗 ，提供了实验工具，遗传和发育研究，包括完全注释的基因组序列，转座子转基因基础，有效的RNA干扰（RNAi）的剧目。在这些优点，基于RNAi的基因敲除技术是在赤研究的核心。T。盗显示出强大的全身性RNAi的反应，使得有可能通过简单地注入双链RNA（dsRNA）的成甲虫的体腔内进行RNAi的任何生命阶段。 在这份报告中，我们提供T。我们的幼虫RNAi技术的概述拟谷盗 。该协议包括T的适当阶段（ⅰ）隔离谷盗幼虫注射，（ii）为在注射设定制备，以及（iii）的dsRNA注射。幼虫RNAi是一种简单，但功能强大的技术，为我们提供了快速访问功能丧失的phenotypES在内的多个基因敲除表型以及一系列亚效等位基因的表型。由于几乎所有的T。赤拟谷盗的组织很容易受到细胞外的dsRNA，幼虫RNAi技术可以让研究人员研究了各种各样的不同背景，包括有机体响应外部环境的遗传基础组织。此外，该技术的简单性刺激在研究多个学生介入，使得T。盗理想的遗传系统，在课堂环境中使用。

一个成功注入的关键步骤，是使一个很好的针。如在步骤6中描述的，需要在针的尖端注入T.之前被破碎拟谷盗 。好的和坏的针的例子示于图1。好的针的尖锐和坚硬尖端，具有0.05mm左右直径的开口（ 图1B）。 图2呈现了一个成功的注射（ 图2A和2B），以及不成功注射（ 图2C-2F）的几种情况。用适当大小的注射针，针尖端穿透幼虫角质层以最小的阻力（ 图2A），并且所述dsRNA溶液（绿色）流入幼虫无任何泄漏（ 图2B）。不要过度注射，因为它会导致双链RNA溶液溢出，甚至用适当大小的注射针（ 图图2C）。如果针尖太薄，针尖常常无法穿透幼虫角质层和弯曲，使得喷射困难的（ 图2D）。如果发生这种情况，修整针尖有时帮助。较大的针头往往仍然可以使用，尽管在穿透角质层幼虫（ 图2E）一些困难。然而，产生大量的dsRNA的溶液易于压出的针，即使在注射器轻微的压力，常常导致dsRNA的溶液（ 图2F）的溢出。 当进行RNA干扰，它有一个阳性对照，以评估该RNAi的工作是否正常，和作为阴性对照，以验证所观察到的效果是不dsRNA的非特异性效应或结果的注射程序（例如损伤引起的是很重要的通过注射，或从醚化异常）。注射双链RNA靶向EYFP可以作为良好的积极控制使用PU-11菌株5,7时。当EYFP的dsRNA是在过去的幼虫阶段喷射中，显著减少将来在翼原基EYFP信号可以作为早一天后喷射（ 图3A）被观察到。 EYFP的击倒是完整的注射的EYFP的dsRNA（ 图3B）的两天后和这个击倒仍然存在通过蛹阶段（ 图3C和3D）和整个甲虫的寿命，如果dsRNA的浓度足够高时（如1微克/微升），7。EYFP的dsRNA也可以被用作阴性对照，因为它是一种非内源基因的序列，应该不会造成形态或生理的干扰（ 图3A-3D）。 我们所提供的RNAi幼虫的残留（VG）的图像，一个关键的基因翼12，作为如何EF另一个例子fective此喷射基于RNAi技术是在T。拟谷盗 。时的dsRNA为VG被注入倒数第二级的幼虫（最后幼虫阶段之前一个阶段），机翼结构的完全丧失所用的蛹期（ 图3E和3F）被观察到。所得的成人也完全缺乏机翼结构（ 图3G和3H）。 RNA干扰结果VG体现了双方的鲁棒性和RNAi在T的系统性拟谷盗 。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52059/52059fig1highres.jpg" width="500" /> 图1:完整的，好，坏针（A）的例子（B）一间破好针尖（C）的断针过大而钝尖（四）提示。的破产<html> Ñ​​针太薄。比例尺（红色）分别为0.05毫米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52059/52059fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52059/52059fig2highres.jpg" width="500" /> 图2:（A）适当大小的注射针插入到末龄幼虫（二）幼虫适当地注入绿色的双链RNA溶液（C）在造成过喷射的喷射点的双链RNA溶液溢出（D ）细针未能穿透角质层幼虫（五）大注射针插入到末龄幼虫（F）幼虫注射用大针头，导致溢出的双链RNA溶液中和泄漏。箭头指示针。ove.com/files/ftp_upload/52059/52059fig2highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52059/52059fig3highres.jpg" width="500" /> 图3:成功的RNAi在T的例子拟谷盗 （AD）的最后幼虫注射EYFP的dsRNA导致减少EYFP表达的。（一）幼虫1天注射后（B）幼虫2天注射后（C）控制蛹（D）从蛹产生幼虫EYFP的双链RNA注射。阴性对照的缓冲注射（A，B）和红色荧光双链RNA注射液（C，D）。箭头和箭头表示EYFP表达或缺乏在边路原基和眼睛分别。（EH）笔最终幼虫的RNAi为VG（E）野生型蛹（F）VG RNAi的蛹。翼结构的缺乏是在蛹期（箭头）。G）的野生型成年。（H） 的vg RNAi的成人已经可见。荣与结构完全缺失（箭头）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52059/52059fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum at JoVE.com

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA干扰（RNAi）技术为基础的基因敲除技术是在赤研究的核心。这里，我们提供了在赤拟谷盗我们的幼虫RNAi技术的概览。幼虫RNAi是一种简单，但功能强大的技术，可以快速访问功能丧失的表型，使研究人员能够研究基因的功能在不同的上下文中。

幼虫RNA干扰在赤拟谷盗，<em&gt;赤拟谷盗</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

larval-rna-interference-in-the-red-flour-beetle-tribolium-castaneum

Universidad San Sebastian

Research

JoVE Journal

Biology

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

16.0K 次观看.  Miami University.  RNA干扰（RNAi）技术为基础的基因敲除技术是在赤研究的核心。这里，我们提供了在赤拟谷盗我们的幼虫RNAi技术的概览。幼虫RNAi是一种简单，但功能强大的技术，可以快速访问功能丧失的表型，使研究人员能够研究基因的功能在不同的上下文中。 

视频: Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

科研

遗传学

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

环境科学

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

发育生物学

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

生物学

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

<meta charset="utf8"></head><body>探索森林蛀木虫的昆虫病原真菌资源

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

<meta charset="utf8"></head><body>作者聚焦：野生 Monochamus alternatus 种群中昆虫病原真菌的评估，用于森林蛀木虫的生物防治应用

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

推进树 突毛 虫 RNAi 技术用于基因功能分析

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

作者聚焦:研究 Wolbachia 诱导的 Thelytokous 孤雌生殖和通过 RNA 干扰的遗传工具包开发

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

幼虫RNA干扰在赤拟谷盗，

摘要

探索更多视频

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

光片为主的生活的荧光显微镜或固定和染色

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

在体外重组活跃T。谷盗端粒酶

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura