饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em>白腹皮蠹</em
Summary
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。
Abstract
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.
Introduction
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。
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>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。 请点击此处查看该图的放大版本。
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。
Protocol
Representative Results
Discussion
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，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.).
Materials
| Dermestes maculatus live beetles | Our lab or Carolina Biological Supply | #144168 | Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out |
| Wet cat food | Fancy Feast | Chunks of meat with gravy. Can buy at most pet food and grocery stores | |
| Dry dog food | Purina Puppy Chow | Can buy at most pet food and grocery stores | |
| Insect cage (size medium, 30.5x19x20.3 cm) | Exo Terra | PT2260 | For colony maintenance. Can use larger cage if needed |
| Insect cage (size mini, 17.8×10.2×12.7 cm) | Exo Terra | PT2250 | For embryo collection |
| Petri dish | VWR | 89038-968 | |
| Cotton ball | Fisher | 22-456-883 | |
| Megascript T7 transcription kit | Fisher | AM1334 | For 40 reactions |
| Pneumatic pump | WPI | PV830 | |
| Capillary holder | WPI | ||
| Micromanipulator | NARISHIGE | MN-151 | |
| Black filter paper (90 mm) | VWR | 28342-010 | |
| Food coloring (green) | McCormick | ||
| Borosilicate glass capillary | Hilgenberg | 1406119 | |
| Needle puller (micropipette puller) | Sutter Instrument Co. | P-97 | |
| Microscope glass slide | WorldWide Life Sciences Division | 41351157 | |
| Sealing film (Parafilm M) | Fisher | 13-374-12 | |
| Model 801 Syringe (10 µl ) | Hamilton | 7642-01 | |
| Needle (32-gauge) | Hamilton | 7762-05 | |
| Fixation Solution (Pampel's) | BioQuip Products, Inc. | 1184C | Toxic, needs to be handled in fume hood |
| Forcep (DUMONT #5) | Fine Science Tools | 11252-30 | |
| Cover slip (24X50 mm, No. 1.5) | Globe Scientific | 1415-15 | |
| Eppendorf Femtotips Microloader pipette tip | Fisher | E5242956003 | |
| Dissecting microscopy for embryo injection | Leica | M420 | |
| Dissecting microscopy for larval phenotypic visualization | Zeiss | SteREO Discover. V12 | |
| DIC microscopy | Zeiss | AXIO Imager. M1 | |
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