Name: rearing and double-stranded rna-mediated gene knockdown in the hide beetle, dermestes maculatus
Uploaded: 2016-12-28T13:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

We thank Drs. Alison Heffer and Yong Lu for setting up the microinjection apparatus and sharing their invaluable knowledge and experience with insect RNAi. This work was supported by the National Institutes of Health (R01GM113230 to L.P.).

1. D.斑的饲养注:D斑的繁殖地使用成人设立在作者的实验室和幼虫购得。使用DNA条形码53种身份进行了验证。 <ol><li>要建立实验室新笼，传播刨花薄薄的一层到一个中等大小的虫笼（30.5×19×20.3厘米3）。将保丽龙的约10×6×3厘米3块在笼子里，让对化蛹的幼虫隐藏。添加20 - 50甲虫（成人或晚龄幼虫）。甲虫将在刨花隐藏。 </li><li>加湿猫粮在培养皿或称量船上。盖上网眼布笼，并把笼子中的孵化器。 </li><li>用于在实验室中保持繁殖地，设置25和30℃之间的温度。 D.斑生长在较高的温度下更快，但是，这也促进了真菌和螨虫的生长。为了维持治疗你的殖民地，使用25 - 28°C进行定期保养的股票和30℃迅速扩大的殖民地。生命周期需要在三周至四个月取决于环境因素50（ 图2）。 </li></ol><img alt="图2" src="/files/ftp_upload/54976/54976fig2.jpg" /> 图 2:D斑 实验室殖民地。一个典型的D.斑虫笼的照片所示。刨花散布让甲虫隐藏。猫的食品在小培养皿或称重舟说。泡沫塑料放入笼最终龄幼虫化蛹的避难所。这里示出的保持架是30.5×19×20.3厘米3和容纳几百幼虫，蛹和成虫。 <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li> LEA鸡蛋已经在笼中走向成熟。为扩大殖民地，如上，幼虫，成虫或（如第2节所述收集），鸡蛋建立新的笼子。 D.纹豆象产卵整个笼子里，特别是靠近食物来源。 </li><li>更换大约一个星期两次湿的猫食。 注:由于湿猫粮的难闻的气味，可替代食物来源也进行了测试（见讨论）。 </li></ol> 2.胚胎收集<ol><li>要建立一个集合，分开，至少50名男性和从殖民地女性50。年轻人是最好的。发生在成年人没有刨花一个小规模的笼子（17.8 X 10.2×12.7厘米3）。添加猫粮在称量船。 </li><li>在开始收集前应仔细检查腾笼换任何存在的胚胎，并删除它们。把一个延伸棉花球入笼，并留下笼在30℃培养箱。适当的时间窗后，棉球将准备胚胎收集。 </li><li>折在半片黑纸建筑（A4尺寸）来创建一个折痕，然后展开。 </li><li>从笼中取出拉长棉花球。从棉球取出成人，并把它们放回笼子。 </li><li>虽然轻轻捏拉伸的棉花球，把它拆开缓缓驶入棉薄丝，让鸡蛋落在黑纸。 注:D.斑胚胎是脆弱的，很难在棉花地看到。他们可以持有，如果棉球过于牢固轻易粉碎。 </li></ol> 3.准备双链RNA <ol><li> DNA模板制备的合成双链RNA <ol><li>运行4 - 使用含有T7启动子的序列在两个5的引物6 50微升的PCR反应&#39;端根据制造商的协议来扩增DNA模板。 </li><li>净化用市售PCR纯化试剂盒，按照制造商的说明PCR产物。 DNA模板的理想最终浓度为≥100纳克/微升。 </li></ol></li><li> 注射缓冲液的制备 <ol><li>制备100ml注射缓冲液（0.1毫的NaH 2 PO 4，5mM的KCl）中。调节pH至6.8用5M NaOH中。 </li><li>商店等分于-20℃。 </li></ol></li><li> 合成双链RNA <ol><li>设置在冰上0.2mL的PCR管的反应，并进行用T7聚合酶，按照生产商的说明在体外转录反应中，使用〜每反应500纳克的模板。 </li><li>在37℃孵育管过夜。 </li><li>消化用DNase DNA模板，按照生产商的说明。 </li><li>加热管至94℃，并在94℃保持3分钟。 </li><li>退火的双链RNA，慢慢由1℃/分钟，直至1小时后达到45℃冷却该管。 注意:步骤3.3.2通过3.3.5可以在热循环仪中进行。 </li><li>乙醇dsRNA的沉淀，加入280微升无RNase水到20微升的反应，并调整到300微升的最终体积。加入30微升的3M醋酸钠（pH5.2）和650微升乙醇。放置在-20℃冷冻试管过夜。 </li><li>在4℃以15,682 xg离心离心30分钟后，洗所述dsRNA沉淀用70％乙醇。干颗粒和20微升溶解注射缓冲液。 </li><li>测量使用分光光度计在260的dsRNA的浓度。理想的浓度为3 - 5微克/微升。通过在90 V上运行5微升在1％琼脂糖凝胶1:25稀释的dsRNA的30分钟检查质量。预期大小的单条带，将容易地看到。 </li><li>存储双链RNA在-20℃或更低的温度。 注:对于每RNAi敲低实验中，包括一个控制的dsRNA，其中预期不会冲击正在研究的过程。 GFP的dsRNA是大多数实验的一个很好的控制。准备并与experime注入控制的dsRNA侧由端ntal双链RNA。 </li></ol></li></ol> 4.微量注射器和微操作机器人的组装注:参见图3。 <ol><li>连接氮气或其它供气气动泵仪器的压力输入。如果一个气动泵不可用，使用50毫升注射器连接于细管施加压力。 </li><li>脚踏开关连接到泵控制弹出压力流量的遥控接口。 </li><li>附加的玻璃毛细管保持器与管泵的排出压力端口。 </li><li>固定在接近解剖显微镜一个显微毛细管持有人。 </li></ol> 5.胚胎RNA干扰注意: 图4示出四纹豆象胚胎的RNAi的流程图。 <ol><li> eRNAi准备 <ol><li>准备一个胚胎收集培养皿的盖子（90毫米直径）。将一张黑色的滤纸到c在盖的内部。把一个标准的显微镜载玻片上的黑纸。 </li><li>稀释到双链RNA注射缓冲适当的浓度。使用2 - 3微克/初始实验微升。始终注射前保持在冰上的dsRNA。 </li><li>添加食用色素1:40稀释的双链RNA和吸管轻轻几次拌匀。 </li></ol></li><li> 收集胚胎注射 注:在25°C，在胚胎细胞核6 - 8小时后产蛋（AEL）向卵子周围53迁移。 10小时AEL 53 -蜂窝胚是8内建立。以确保前蜂窝胚期胚胎用于注射，0 - 3小时AEL胚推荐使用53。胚胎收集，准备和注射通常需要不到2小时，如果玻璃毛细管都处于良好状态。因此，整个过程可在5小时AEL内完成。 <ol><li>如步骤收集胚胎2.2-2.5。 </li><li>点击黑色建筑用纸胚胎转移到收集培养皿的盖子。 </li><li>粘双面胶带沿着滑动的边缘。 </li><li>使用油漆刷，对准磁带上的胚胎垂直于与在边缘的前或后端滑块边缘。需要注意的是早期胚胎的前部和后部端部不容易区分，因而平均的一半将在后端这是理想的注入。 </li></ol></li><li> 加载双链RNA进入玻璃毛细管 <ol><li>占用2 - 4微升的dsRNA成20微升微加载的枪头。 </li><li>插入的尖端进入预拉玻璃毛细管（称为针），并轻轻地吸取所述dsRNA溶液注入毛细管中。从准备使用微量拉马商用玻璃毛细管针。一种理想的拔出针的一个例子示于图5。长度和针的锥形可能需要被对焦优化之三注射试验。 </li><li>固定玻璃毛细管导入玻璃毛细管保持器。 </li><li>组装毛细管持有人到显微。 </li></ol></li><li> 双链RNA注射胚胎成 <ol><li>小心地用胚胎滑动转印到解剖显微镜的阶段。 </li><li>移动滑板带来一个胚胎到外地和焦点显微镜的中心。 </li><li>而寻找到解剖显微镜毛细管的尖端与显微定位。带来附近的行中的第一个胚胎的端部的尖端。 </li><li>交换机上的氮气或其它气源。 </li><li>设置电磁输入选择开关真空。 </li><li>调整弹出调压器10 - 15磅。调整取决于该装置的压力。 </li><li>如有必要，打开毛细管。一旦该尖端部是开放的，有色的dsRNA溶液将填补小费。 </li><li>向前移动毛细管尖端穿刺电子商务mbryo。如果压力设置是理想的，无胚胎的流体将流入所述毛细管。 </li><li>步骤脚踏开关上以喷射的dsRNA溶液进入胚胎直到溶液适量喷出。 图5示出的溶液适当和不适当的量被注射后胚胎形态的例子。 </li><li>保持毛细管尖端胚胎的〜2秒内，然后将其删除。 </li><li>移动毛细管到下一个胚胎，并重复步骤5.4.8 - 5.4.10，直到所有的胚胎注入。更换玻璃毛细管，如果它被堵塞或破裂。 </li></ol></li><li> 注射后恢复和培养 <ol><li>注射后，放置在培养皿中的幻灯片。 </li><li>添加湿棉球培养皿。不要让棉花球碰的幻灯片。 </li><li>盖培养皿中，用封口膜包裹。标签的盘用的dsRNA，浓度，日期，时间，和数量的注入的名称胚胎。 </li><li>放置在30℃的培养箱中，直到胚胎孵化培养皿。 </li></ol></li></ol> 6.家长的RNAi <ol><li> 隔离蛹为pRNAi <ol><li>从殖民地收集蛹。 </li><li>排序男性并在显微镜下雌性蛹（ 见图6）。保持它们在30℃的培养箱中两个单独的培养皿，以确保在注射前没有配合发生。 </li><li>检查每天eclosed成年人。化蛹通常需要5 - 7天30℃。 </li><li>转让eclosed男性和女性（ 见图6），以两个独立的培养皿中，并喂它们猫粮隔日，直到他们准备注射。 </li><li>继续注射，一旦有足够多的女性，并在适当的年龄男性。女4〜8天后羽化是最好的。对于一个典型的分析，8 - 使用12位女性。都需要配合54男性数目相等。 </li></ol></LI&gt; <li> 双链RNA注射到雌性腹部 <ol><li>冰（5.1.2和5.1.3）准备的dsRNA。 </li><li>附加32号针头至10微升的注射器。 </li><li>麻醉对CO 2阶段的女性。 </li><li>加载的dsRNA溶液到注射器而不占用任何空气。 </li><li>举办女腹侧用一只手，并与其他注射器。 </li><li>轻轻穿透腹板2和3之间的segmacoria（膜）与针的尖端。 图7示出在注射过程中女性。如果针不容易穿透组织，角度针向上和慢慢向下压。插入针的大约2mm到体内。 </li><li>检查注射器刻度一边慢慢推柱塞，注射每雌〜2微升。 </li><li>注射后，从女性的腹部取出前保持针仍然为至少5秒。 </li><li>转移注入雌性至培养皿（直径90mm），并用猫食饲料。 </li><li>标签的培养皿所述dsRNA名称，注射量，日期，和女性的数目。 </li></ol></li><li> 注射后恢复和配合 <ol><li>孵育在30℃的培养箱中的培养皿。 </li><li> 24小时后，转移注入雌性到迷你笼。 </li><li>添加到笼子未注射年轻男性的数量相等，并贴上标签笼。 </li><li>添加与猫的食物称重船入笼。 </li><li>离开笼在30℃的培养箱中，以允许配合。 24小时后，女性应开始产卵和胚胎可以每日或在所需的时间间隔来收集。 </li></ol></li></ol> 7.表型分析后的RNAi 注:在30°C，它需要〜55小时的鸡蛋孵化50。 <ol><li> eRNAi生存力分析 <ol><li>通过孵出幼虫的数目进行比较，以总NU计算孵化率注射胚胎的MBER。一定要包括阴性对照作为注入胚胎可能受到伤害或注射过程杀害。 60％ - 典型的孵化率为30％而有所不同。谨防孵幼虫吃未孵化的蛋。 </li></ol></li><li> pRNAi生存力分析 注意:如果女性生存能力是通过RNAi靶向基因的影响，女性注射特定的双链RNA，但不是阴性对照双链RNA就会开始死亡。如果产蛋或产蛋受到影响，女性会表现出部分或完全无菌。相对于阴性对照评分女性的生存或通过比较实验和控制女性（7.2.3）产卵的总数。 <ol><li>接下来的步骤2.2建立胚胎收集。 </li><li> 24小时后，收集以下步骤2.4胚胎 - 2.5。 </li><li>算胚胎的总数。 </li><li>胚胎转移至90毫米的培养皿。标签培养皿有针对性的基因，采集日期和胚胎数。 </LI&gt; <li>孵育胚胎在30℃的培养箱中直到孵化。由于对未孵化的胚胎孵化出仔鱼饲料，使用镊子（见讨论）从胚胎未孵化的检查培养皿经常和独立孵幼虫。 </li><li>计数孵出的胚胎的数目和计算孵化率。 </li></ol></li><li> 检查角质层缺陷孵幼虫 <ol><li>在1.5 ml离心管收集孵幼虫。 </li><li>在通风橱中，加入1 ml固定溶液中。 </li><li>离开，在4℃下的离心管中至少过夜以让该溶液渗透幼体组织。 </li><li>可视化，从管中删除尽可能多的固定液。 </li><li>漂洗幼虫至少三次用PBST。 </li><li>转移幼虫与使用PBST与端的P 1000枪头多井玻璃板切断扩大它。 </li><li>在解剖显微镜下，使用镊子来检查缺陷舒展幼虫。一世t是伸出幼虫检查缺陷固定液他们的身体合同的关键。 </li></ol></li><li> 检查角质层缺陷未孵化的幼虫 注意:此过程是早期胚胎表型的考试有用的，尤其是对于那些不求生存，以孵化的胚胎。 <ol><li>为pRNAi，添加PBST入培养皿（7.2.4）和利用P-1000枪头未孵化的胚胎转移到1.5ml微量离心管中与端切断。对于eRNAi，加入PBST到培养皿（5.5.4），刷未孵化的胚胎关闭显微镜载玻片，并将它们转移到一个离心管中。 </li><li>修正了固定液的胚胎，并用PBST冲洗一下可视化下面的步骤7.3.2 - 7.3.6。 </li><li>使用镊子，解剖胚胎出蛋壳的在PBST在解剖显微镜下。 </li><li>胚胎转移回1.5 ml离心管（〜200μl的胚胎在每个管）。 </li><li>删除尽可能多的溶胶ution越好。 </li><li>加入1ml 90％的乳酸/ 10％EtOH中。留在60℃的培养箱离心管至少过夜。 注:胚胎可以在60℃下放置数天。 </li><li>挂载胚胎，利用P-1000枪头与端切断吸取他们到显微镜载玻片。 </li><li>用钳子将胚手动定位到一个理想的方向。 </li><li>盖上盖玻片胚胎，并使用DIC显微镜下可视化。 </li></ol></li><li> 检查细胞与分子缺陷 注:此过程对于调查未伴有表皮角质层的缺陷或表型杀伤力的根本原因很有用。 <ol><li>对于pRNAi，从棉球独立的胚胎在适当的发展阶段，并将它们转移到一个胚胎收集筐。收集筐可以是相同或类似的用于果蝇胚胎集合。 <ol><li>使从去除小瓶的底部25毫升塑料闪烁小瓶蛋篮，和一个圆切口盖（大约直径为1厘米）的。地方超细一圈网状约2.5厘米的盖子内径，然后拧上闪烁瓶中，用颠倒。 </li><li>作为网眼可以一次将胚洗涤容易地除去，浸入网格在一个离心管中与水以回收附着在网状任何胚胎。 </li></ol></li><li>为eRNAi，在适当的阶段，添加PBST到培养皿。刷过的胚胎在显微镜载玻片，并将它们转移到一个胚胎收集筐。 注:固定，原位杂交，抗体染色和核染色协议可以在Xiang 等中找到。 2015年53。 </li></ol></li></ol>

The authors have nothing to disclose.

虽然在20 世纪期间，开发了先进的模型系统（老鼠，苍蝇，蠕虫）的一小部分，21 世纪已经出现在世界各地的实验室正在开发新的动物系统的浪潮。这些新系统让科学家来解决不能只使用"标准"模式的系统可以探测比较，进化的问题。新车型这一部署要求，为培养实验室，基因鉴定，并在新种功能的方法方法的快速发展。这里，分别提出了在实验室和一步一步协议胚胎的RNAi，父母...

在1998年，消防和Mello报道，双链RNA（dsRNA）的可诱导基因功能的抑制线虫 1。通过双链RNA触发此响应被命名为RNA干扰（RNAi），并且据报道这种RNAi介导的基因沉默在动物，植物和真菌2-7是保守的。在植物和一些动物，RNA干扰的功能全身的，这意味着影响可以传播到的dsRNA不直接导入其它细胞/组织（在8-10中综述）。科学家利用通过设计的dsRNA的目标感兴趣的基因这种内源性细胞RNAi应答，从而击倒基因功能，无需直接操纵基因组（11-14综述）。 RNA干扰是由于以下优点功能研究的强有力的工具。首先，甚至以最小的基因序列信息，一个基因可以通过使用RNA干扰的目标。这是圣特别重要缺乏的基因组或转录组数据的非模式生物udies。第二，在生物体，其中所述RNAi响应是鲁棒全身性的，RNAi介导的基因敲除，可以在几乎任何发育阶段进行。此功能是用于研究多效基因的功能非常有用的。第三，在一些情况下，RNA干扰效果扩散到性腺和后代，使得表型在后代15,16观察到。这种现象，被称为亲RNA干扰（pRNAi），为基因影响胚胎发育特别有利的，因为由单个注射亲本产生的大量后代可以不鸡蛋的直接操作进行检查。由于这些原因，pRNAi是选择的方法。然而，如果pRNAi是无效的，例如用于对卵子发生所需的基因，那么胚胎RNA干扰（eRNAi）必须使用。第四，RNAi技术可以用来产生在一个等位基因系列dsRNA的量交付可以在一定范围内变化的等效，以产生弱到强的缺陷。表型的这种灰度可以是用于理解基因功能，当基因参与一个复杂的过程和/或功能的完全丧失是致死有帮助的。第五，dsRNA的交付通常是很容易的，可行的，尤其是在动物显示出强大的系统性的RNAi反应。双链RNA可以通过显微注射1,5，喂食/ 17,18摄入，浸泡，19,20和病毒/细菌介导的递送21,22引入。第六，不像一些基因打靶/编辑方法，没有必要以筛选携带突变生物体或开展遗传杂交使用的RNAi时生成纯合子。因此，与许多其他技术用于研究基因功能，RNAi是快速，廉价，并且可以应用于大型屏幕23-25。 RNAi技术的广泛实用程序提供了手段，广泛的生物进行功能研究，扩大品种范围可供研究博扬ð为其遗传工具已经开发的传统模式系统。例如，使用非模型系统的研究是必需由种代表不同的发展模式，或表现出不同的形态特征比较26-29同源基因的功能给予见解的基因和基因网络的演进。这些类型的研究将提供一个更好的理解生物多样性的，与两个应用和基础研究的影响。 作为地球上最大的动物群，昆虫提供了一个很好的机会，探讨其作用机制基本多样性。此外，昆虫一般都比较小，生命周期很短，繁殖力高，而且很容易在实验室后方。在过去的二十年中，RNA干扰已成功应用于昆虫跨越的订单，其中包括双翅目（真苍蝇）5，鳞翅目（蝴蝶和飞蛾）30，鞘翅目（甲虫）16,31，膜翅目（声表面波滤波器谎言，黄蜂，蚂蚁和蜜蜂）32，半翅目（真正的错误），等翅目（白蚁）34，蜚蠊目（蟑螂）35，直（蟋蟀，蚱蜢，蝗虫，螽斯和）36 Phthiraptera（虱子）37。 RNA干扰的成功应用，提供了用于在早期胚胎图案形成的研究功能数据（前后轴32，背腹轴28，分割26,38），性别决定39,40，壳多糖/角质层生物合成41，蜕皮激素信令42，社会行为43，等等。对于不同的昆虫物种开发RNAi的方法可能的，因为它们很可能是对虫害控制（在44-46综述）有用的附加的好处。 RNAi的影响将是基因特异性以及种特异性，只要非保守区被选择用于靶向。对于益虫物种，如蜜蜂和蚕，目标基因的生存至关重要病毒或寄生虫感染控制可提供保护这些物种47,48的新策略。 白腹皮蠹 （ 四纹豆象 ），俗名隐藏的甲虫，是分布在世界各地，除了南极洲。作为holometabolous昆虫中，D.斑生命周期包括胚胎，幼体，蛹和成虫阶段（ 图1）。因为它的肉源，D.斑被博物馆用来缩略动物尸体和法医昆虫学家可以用它来估计死亡49,50的时间。 D.斑饲料对动物产品，包括尸体，风干肉，奶酪和蛹/其他昆虫的茧，从而造成损害的家庭，储存食物，丝绸，奶酪和肉类行业51,52。在这种甲虫应用RNAi技术可以提供一个高效和环保的方式，以尽量减少其经济影响。我们的实验室使用了斑D.作为一个新的M奥德尔昆虫研究的分割53。除了是服从实验室饲养，D.纹豆象是基础研究的兴趣，因为它是一个中间胚芽开发，使之成为一个有用的物种，研究短期和长期牙胚发育之间的过渡。 <img alt="图1" src="/files/ftp_upload/54976/54976fig1.jpg" /> 图1:D斑的生命周期。 D.斑的照片在不同的生命阶段，如图所示。从卵到成年生命周期需要在30℃C三星期，但不再在较低温度下。 （A，F）刚打下胚胎白色至浅黄色和椭圆形，约1.5毫米长。胚胎发育需要〜30℃下55小时。 （B，C和G）幼虫有深色色素条纹和覆盖着刚毛。幼虫通过根据环境和其长度几龄可以延伸到超过1厘米。 （D，H） </str翁&gt;年轻蛹是浅黄色。在30°C 7天 - 化蛹，大约需要5。 （E，I）羽化后不久，深色素沉着出现在成虫体。成人能活到数月，一名女性能在她的一生打下数百胚胎。 <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 以前，我们发现，RNAi是有效的，在D. 53 斑撞倒基因功能。这里我们的经验饲养D.斑菌落在实验室用一步一步协议胚胎和亲RNAi的设置，注射，注射后护理，和表型分析沿共用。这里介绍的dsRNA介导的基因敲低和分析方法不仅为D.斑寻址问题提供详细的信息，但也有FO潜在意义R IN其他非模型甲虫/昆虫RNAi的应用。

<table><tbody><tr><td>&lt;em&gt;Dermestes maculatus&lt;/em&gt; live beetles</td><td>Our lab or Carolina Biological Supply</td><td>#144168</td><td>Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out</td></tr><tr><td>Wet cat food</td><td>Fancy Feast</td><td></td><td>Chunks of meat with gravy. Can buy at most pet food and grocery stores</td></tr><tr><td>Dry dog food</td><td>Purina Puppy Chow</td><td></td><td>Can buy at most pet food and grocery stores</td></tr><tr><td>Insect cage (size medium, 30.5 x 19 x 20.3 cm)</td><td>Exo Terra</td><td>PT2260</td><td>For colony maintenance. Can use larger cage if needed</td></tr><tr><td>Insect cage (size mini, 17.8 x 10.2 x 12.7 cm)</td><td>Exo Terra</td><td>PT2250</td><td>For embryo collection</td></tr><tr><td>Petri dish</td><td>VWR</td><td>89038-968</td><td></td></tr><tr><td>Cotton ball</td><td>Fisher</td><td>22-456-883</td><td></td></tr><tr><td>Megascript T7 transcription kit</td><td>Fisher</td><td>AM1334</td><td>For 40 reactions</td></tr><tr><td>Pneumatic pump</td><td>WPI</td><td>PV830</td><td></td></tr><tr><td>Capillary holder</td><td>WPI</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>NARISHIGE</td><td>MN-151</td><td></td></tr><tr><td>Black filter paper (90&amp;nbsp;mm)</td><td>VWR</td><td>28342-010</td><td></td></tr><tr><td>Food coloring (green)</td><td>McCormick</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>Hilgenberg</td><td>1406119</td><td></td></tr><tr><td>Needle puller (micropipette puller)</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Microscope glass slide</td><td>WorldWide Life Sciences Division</td><td>41351157</td><td></td></tr><tr><td>Sealing film (Parafilm M)</td><td>Fisher</td><td>13-374-12</td><td></td></tr><tr><td>Model 801 Syringe (10&amp;nbsp;&amp;micro;l)</td><td>Hamilton</td><td>7642-01</td><td></td></tr><tr><td>Needle (32-gauge)&amp;nbsp;</td><td>Hamilton</td><td>7762-05</td><td></td></tr><tr><td>Fixation Solution (Pampel&#039;s)</td><td>BioQuip Products, Inc.</td><td>1184C</td><td>Toxic, needs to be handled in fume hood</td></tr><tr><td>Forcep (DUMONT #5)</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Cover slip (24 x 50 mm, No. 1.5)</td><td>Globe Scientific</td><td>1415-15</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader pipette tip</td><td>Fisher</td><td>E5242956003</td><td></td></tr><tr><td>Dissecting microscopy for embryo injection</td><td>Leica</td><td>M420</td><td></td></tr><tr><td>Dissecting microscopy for larval phenotypic visualization</td><td>Zeiss</td><td>SteREO Discover. V12</td><td></td></tr><tr><td>DIC microscopy</td><td>Zeiss</td><td>AXIO Imager. M1</td><td></td></tr></tbody></table>

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.

作者的实验室已用RNAi技术来研究基因调节昆虫53,55分割功能的进化。虽然所有的昆虫被分割，调节这一过程的基因出现在昆虫辐射26,38,56-63有分歧。在果蝇遗传筛选确定了一组九个是负责促进主体段64-70的形成一对规则分割基因。这里，这些基因中的一个的直向同源物， 成对 （PRD），用于记录的RNAi的用途为在D斑研究基?...

Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

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

在这里，我们提出了在实验室饲养的中间胚芽甲虫， 白腹皮蠹 （ 四纹豆象 ）协议。我们还同胚胎和父母的RNAi方案和方法分析表型的胚胎基因功能研究该物种。

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 ...

rearing-double-stranded-rna-mediated-gene-knockdown-hide-beetle

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

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

10.6K 次观看.  University of Maryland. 在这里，我们提出了在实验室饲养的中间胚芽甲虫， 白腹皮蠹 （ 四纹豆象 ）协议。我们还同胚胎和父母的RNAi方案和方法分析表型的胚胎基因功能研究该物种。 

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

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 ...

科研

环境科学

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

神经科学

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

奥多纳塔电介质介质RNA干扰法

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

发育生物学

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

RNA干扰在美洲蟑螂中的应用

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

生物学

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

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 ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

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.

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 ...

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

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通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus

在大规模的基因敲除

幼虫RNA干扰在赤拟谷盗，

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

RNAi的触发传送到