The authors would like to thank all the members of the "Insect Genetics and Genomics" Laboratory, in particular to Lorenzo Ghiringhelli who has worked at developing, adapting and maintaining the rearing of the medfly over the past thirty years. Part of the representative results of this paper have been reprinted from N. Biotechnology, 25(1) by Scolari F. et al., Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae), 76-84, 2008, with permission from Elsevier (License number 3796240759880). This work received support from Cariplo-Regione Lombardia "IMPROVE" (FS).

1.实验装置<ol><li> 养虫要求 <ol><li>维护所有C.实蝇生命阶段在25°C，湿度65％和12/12小时的光/暗光周期。 </li><li>将约1,500-2,000地中海果蝇蛹在6升笼子。使用笼黄铜一侧网有孔小到足以刺激产卵42。插入海绵​​条通过在笼的底部的小开口通过毛细作用的手段，以提供与水蝇。使用酵母和糖（1:10）作为用于成年人（ 图1A）的食物源的混合物。提高酵母的量（高达1:3，酵母:糖）应女性不产卵。 </li><li>将充满水笼下方的板收集通过网格沉积在鸡蛋。随着处女雌可以排卵，等到苍蝇是收集卵子显微注射实验先老，至少6-7天。这将确保女性都会有交配并且该鸡蛋将已受精。 </li><li>通过用细网过滤器过​​滤的水收集鸡蛋。从过滤器转移蛋在塑料盒含有标准幼虫食物（1.5 LH 2 O，加入100ml盐酸1％，5克的广谱抗微生物剂溶解在50毫升乙醇中400克糖，175克软化水啤酒酵母，1使用巴斯德吸管公斤软麦麸）。 </li><li>放置每个方块在用网盖关闭的透明容器，让空气流通，并含有麸皮层有利于化蛹（ 图1B）。 </li><li>经过约10天，检查3 龄幼虫，从幼虫的食物出现并且跳进了化蛹麸皮层。 </li><li> 24小时后，用软刷收集了一小杯蛹并把它们转移到一个新的成人笼子。正常成人化蛹后10天出现。新兴飞使用ptilinum，充气袋位于其头顶次触角电子基，以强制关闭puparium的末尾。出苗后，ptilinum永久性崩溃后脑勺里面。 </li></ol></li><li> 1升食品准备显微注射幼虫 <ol><li>溶解2.5克琼脂在300毫升H 2 O </li><li>温馨500毫升H 2 O于热搅拌盘和化解4克苯甲酸钠，4.5毫升37％的盐酸，41克酵母提取物和115克脱水胡萝卜粉。 </li><li>添加到混合物溶于10毫升无水乙醇2.86克广谱抗微生物剂的溶液中。 </li><li>分发媒体15厘米的圆形培养皿并冷却。如果有必要，应储存于4℃。 </li></ol></li><li> 幻灯片准备 <ol><li>放置双面胶带2厘米长条在显微镜载玻片（75×26 平方毫米）和标记有中国标记的边缘，以防止油扩散和注入胚胎的随后干燥。 </li></ol></li><li><st荣&gt;核酸制品<ol><li>使用市售的试剂盒34分离质粒DNA。用乙醇沉淀质粒，并在注射缓冲液（0.1 mM磷酸盐缓冲液pH 7.4，5mM的KCl）中在所需的浓度重新悬浮。 </li><li>纯化在体外使用苯酚-氯仿原料合成长的dsRNA，沉淀用异丙醇和在注射缓冲液43重新悬浮。 </li></ol></li></ol> 2.胚芽制备<ol><li>将含有6-7日龄成虫笼下一个装满水的托盘。 </li><li>允许雌性产卵30分钟，然后通过使用一个细滤网过滤水收集胚胎。始终保持滤网的水，以避免胚胎干燥。 </li><li>通过浸没式为5秒的过滤器在商业50％漂白溶液Dechorionate胚胎。 </li><li>通过反复佛光普照清洁超纯水过滤（至少仔细清洗胚胎4-5倍）。最后，将干净的超纯水过滤。使用最多2小时内的胚胎。 注:显微注射的定时相关的必要性时细胞化之前核分裂的相位以注入胚前期胚胎，。这允许将要采取行动的注射DNA导入细胞核，具体到原始生殖细胞的细胞核，将发起的配子。在果蝇中 ，细胞化在极细胞开始在约90分钟受精后（在25℃），具有胚形成中发生约30分钟后44，而在地中海果蝇细胞化需要9和12小时45,46之间。 </li><li> 行胚胎方向 <ol><li>使用精细的画笔，收集约50胚胎，并把它们用清水浸泡黑色滤纸的磁盘上。 </li><li>在解剖立体显微镜，安排在一排的胚胎在双面胶带的上表面上用细画笔（000）的显微镜载片（ 图1C）。 注:对齐都在同一个方向的胚胎，作为喷射在后极，其中，种系将发起执行;后极是相反的珠孔区域。 </li><li>盖上三氟了一层油确保油不溢出中国的标记边界（见1.3）的胚胎。 注意:覆盖油胚胎之前，可以执行进一步的步骤:胚胎可以干燥处理几分钟，以减少它们的液体含量，从而促进注入的DNA溶液的入口。这可以帮助避免注射或注射溶液的过量泄漏期间胚胎破裂，因为高的液体压力的结果。 </li></ol></li></ol> 3.胚胎显微注射<ol><li>填充针（1-2微升）用质粒DNA或用微量的dsRNA溶液（1-2微克/微升）。 </li><lI&gt;针连接到微注射装置，其控制喷射时间（0.5秒），压力（800百帕）和背压（150帕斯卡）。 </li><li>在立体显微镜下进行的显微注射，使用显微来控制针（ 图1D）的运动。使用显微镜舞台上移动的幻灯片。 <ol><li>广场上的范围阶段的幻灯片。 </li><li>插入针的尖端进入胚胎（ 图1E）的后极。 </li><li>通过激活显微注射系统与踏板注入该溶液。注入的液体引起的内压增加，从而导致胚胎的轻微运动。 </li><li>松开踏板，马上，而是轻轻，取出胚胎针。 </li><li>观察注射部位稍微细胞质泄漏。 注:由于注射亲DNA /双链RNA / siRNA和死亡率的潜在致命影响区分cedure本身，对照实验必须执行。这些包括缓冲器的注入，或仅在反向遗传学实验的情况下，绿色荧光蛋白（GFP）的衍生双链RNA（dsRNA的-GFP），或siRNA。 </li></ol></li><li>注射后孵育胚胎在25℃。 </li></ol> 4.后喷射过程<ol><li>注射后两天，查体视显微镜下孵幼虫（ 图1F）的幻灯片。幼虫可在油中移动，但需要被尽快转移到幼虫的食物。为此，检查幻灯片，每天数次。 </li><li>使用精细的画笔（000），在孵幼虫轻轻地转移到基于胡萝卜幼虫的食物。传输最高可达每每个培养皿200幼虫。 </li><li>孵育幼虫在65％的湿度（25℃）。确保盖子不沾的菜，影响空气交换。 </li><li>保持培养皿的盖大多关闭，使得仍有空气流的幼虫。为了避免过度干燥，每天检查的菜，如果需要，加水以补偿蒸发。 </li><li>放置培养皿中在由网状盖关闭明确容器和含有麸皮层有利于化蛹。至于日常的养虫室饲养，3 龄幼虫跳出幼虫的食物和化蛹在麸皮。 </li><li>收集蛹，并放置在一个小笼子成人饲养和筛选（这是第0代，G0）。后在标准幼虫食后代。 </li><li>在种系转化实验的情况下，为您在下一代可能改变个人（G1），作为注入个人通常是嵌合体。换位事件，事实上，可能发生在任何胚胎合胞体的核，这将形成任一种系或体细胞的。 <ol><li>出苗后不久，使用二氧化碳麻醉G1个人和检查TRANSF落射荧光立体显微镜下ormation事件。为了筛选红色荧光的存在，可以使用一个DsRedwide滤波器（内线一十二分之五百四十六;电解金属锰七十五分之六百〇五），而到屏幕为绿色荧光使用EYFP滤波器（内线二十○分之五百;电解金属锰三十〇分之五百三十五） 。 </li><li>将转化个人在一个小笼子里与异性的野生型成人和发起新的初始杂，应变。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

核酸在昆虫胚胎显微注射是一个普遍的技术，该技术有利于基因的功能和生物技术应用中的两个分析。 最近从越来越多的昆虫物种的基因组序列的出版物导致为未知功能的基因的功能表征工具的迫切需要。 RNA干扰已被证明是最有价值的方法之一来推断分子功能49和胚胎显微注射利于这些研究。 质粒DNA注射可用于修改使用转座介导的基因插入昆虫?...

地中海果蝇（地中海果蝇） 地中海实蝇是一个世界性的物种，广泛破坏水果栽培作物。它属于实蝇科家族，包括若干害虫种类，如那些属于属实蝇和实蝇 。在地中海果蝇是这个家庭的研究最多的物种，它不仅已成为昆虫入侵1的研究，同时也为优化虫害治理策略2的模式。 在地中海果蝇是一种多化性品种可以攻击300余种野生和栽培植物3,4。该损害是由成年人和幼体阶段既造成:交配雌刺穿水果的表面产卵，从而使微生物影响他们的商业品质，而在果肉幼虫饲料。经过三年幼虫阶段，幼虫从主机出现，化蛹成土壤。 实蝇结球甘蓝显示一个几乎全世界发行，包括非洲，中东，澳大利亚西部，中美洲和南美洲，欧洲，和美国5的领域。 最常见的策略，以限制地中海果蝇虫害包括使用杀虫剂（ 如马拉硫磷，多杀菌素）和环境友好型昆虫不育技术（SIT）6。后一种方法涉及释放到几十万暴露进行消毒，以电离辐射的男性的野性。这样灭菌的男性野生雌性交配导致没有后代，导致种群大小的减少，最终导致消除。虽然坐在多个广告系列已被证明有效的全球，它的主要缺点包括饲养和灭菌数以百万计的昆虫被释放的高成本。发布的个人标记是必要区分期间在现场捕获的野生昆虫无菌监测活动，它是使用荧光粉末目前实现的。这些过程是昂贵的，并有不希望的副作用7。 为了优化和/或制定这种害虫的控制更加有效的办法，地中海果蝇生物学和遗传学已广泛被全球众多研究人员探讨。在地中海果蝇基因组序列8,9的可用性，将有利于对基因功能的新的调查。 RNA干扰是这类研究的强有力的工具，它可以通过双链RNA（双链RNA）或siRNA（小干扰RNA）的显微注射来实现。这一技术已被使用，例如，以证明在C中的性别决定分子级联实蝇仅部分相对于该果蝇 10的保守的。 协议的发展microinject允许C.地中海果蝇胚胎实蝇是第一非-Drosophilid蝇种进行遗传修饰。作为它的卵是相似的果蝇的，无论是在形态和性方面对干燥11中 ，协议递送质粒DNA到第一对D.开发胚前期胚胎黑 12,13 最初适合用于C.实蝇 。这些第一实验允许基于转座子米诺斯11地中海果蝇种系改造。接着，将原来的系统是使用其他基于转座子的方法改性14。这是piggyBac转从鳞翅目夜蛾 15的情况。该协议已经被进一步优化，这已经允许的其他品种tephritid 16-21的转型也有许多其他双翅目22-31的。所有这些系统依赖于使用一个典型的二元矢量/辅助质粒转化体系:人为的，有缺陷的TRANSPO含有目的基因的儿子被组装到质粒DNA并通过提供转座酶32集成到昆虫的基因组中。已经产生了许多转基因地中海果蝇线，具有多种功能，包括携带诱导杀伤力有条件的显性致死基因的菌株，株产仅限男性后代，因此不需要额外的性别鉴定的策略，并用荧光精子的菌株，这可以提高精度的SIT监测阶段33-37。虽然在转基因生物的野生释放在导频测试已发生针对蚊子仅38,39，至少一个公司正在评估一些转基因地中海果蝇菌株对它们在字段40使用。 胚胎显微注射也可以偏向的新的基因组编辑工具，例如转录活化剂 - 样效应核酸（TALENS），群集定期相互间隔短回文重复序列的发展（CRISPR）/ CRISPR相关蛋白9核酸酶（Cas9）和归巢核酸内切酶基因（HEGs），这将使新的进化和发展的研究，以及扩大现有的生物技术工具箱。基因组编辑的办法已经允许在蚊子的基因41驱动系统的产生，和他们转移到地中海果蝇是迫在眉睫。在这里，我们描述了在显微注射胚胎地中海果蝇核酸可以为上述所有应用程序非常有用的通用协议。

<table><tbody><tr><td>1 x injection Buffer</td><td></td><td></td><td>0.1 mM phosphate buffer pH 7.4, 5&amp;nbsp;mM KCl</td></tr><tr><td>Construct Plasmid</td><td></td><td></td><td></td></tr><tr><td>Helper Plasmid</td><td></td><td></td><td></td></tr><tr><td>dsRNA</td><td></td><td></td><td>Phenol-Chloroform purified</td></tr><tr><td>Standard Larval food</td><td></td><td></td><td>1.5 L H&lt;sub&gt;2&lt;/sub&gt;O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent used in pharmaceutical products dissolved in 50&amp;nbsp;ml of ethanol, 400 g sugar, 175 g demineralized brewer&#039;s yeast, 1 kg soft wheat bran</td></tr><tr><td>Carrot Larval Food</td><td></td><td></td><td>2.5 g agar, 4 g sodium benzoate, 4.5 ml 37% HCl, 42 g yeast extract, 115 g carrot powder, 2.86&amp;nbsp;g broad-spectrum antimicrobial agent , water to 1 L</td></tr><tr><td>Adult Food</td><td></td><td></td><td>yeast extract and sugar (1:10)</td></tr><tr><td>Microscope slides</td><td>Sigma-Aldrich</td><td>Z692247</td><td></td></tr><tr><td>Injection needles&amp;nbsp;</td><td>Eppendorf</td><td>5242956000</td><td></td></tr><tr><td>Microloaders</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Double slided tape</td><td></td><td></td><td></td></tr><tr><td>Whatman Black circle paper</td><td>Sigma-Aldrich</td><td>Z695505</td><td></td></tr><tr><td>Bleach</td><td></td><td></td><td>Diluite 1:2 before use</td></tr><tr><td>Paintbrush (000)</td><td></td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MN-153</td><td></td></tr><tr><td>Microinjector</td><td>Eppendorf</td><td></td><td>Femtojet</td></tr><tr><td>Adult cages</td><td></td><td></td><td></td></tr><tr><td>Chlorotrifluoroethylene oil</td><td>Sigma-Aldrich</td><td>H8898</td><td></td></tr><tr><td>&lt;em&gt;Ceratitis capitata&lt;/em&gt;</td><td></td><td></td><td>The strain used is ISPRA</td></tr></tbody></table>

delivery of nucleic acids through embryo microinjection in the worldwide agricultural pest insect, ceratitis capitata

地中海果蝇（地中海果蝇） 地中海实蝇 （威德曼）（双翅目:实蝇科）是一种害虫具有极高的相关性农业。这是由于其繁殖行为:女性损坏的水果和蔬菜的外表面时，他们产卵和他们的纸浆孵仔鱼饲料。野生C.实蝇种群是通过传统的杀虫剂喷洒控制和/或环保的方法，最成功的是昆虫不育技术（SIT）。到SIT依赖于大规模饲养，基于辐射灭菌，并且保持它们交配能力，但不能够生成可育后代的男性字段释放。的出现和生物技术工具在随后的快速发展，与地中海果蝇基因组序列的可用性在一起，极大地增强了我们这一物种的生物学的理解。这种青睐的新战略增殖基因组操纵，其中CAN为适用于控制人口。 在这种情况下，胚胎显微注射对扩大工具箱为地中海果蝇控制的双重角色。与该调节键的生物过程的基因的功能，以干扰的能力，事实上，扩展了我们的分子机械地中海果蝇侵袭下层的理解。另外，为了达到种系转化的能力有利于生产，可以在新的SIT设置将来的现场应用进行测试的多个转基因品系。事实上，基因操作可以用来赋予能，例如，可以使用在该领域并监控育雄性能理想性状，或可能导致生命早期阶段致死。这里，我们描述的方法来microinject核酸导入地中海果蝇胚胎来实现这两个主要目标。

在这里，我们分别报告指示在感兴趣的基因的功能表征胚胎显微注射的两个应用程序（情况1）中，在（情况2）的代转基因品系的。 dsRNA的交付成胚胎解开基因的功能。 所述innexin-5基因编码，在昆虫，具体在雄性和雌性性腺43,47,48表示的间隙连接。根据可用于密切相...

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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. ...

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

9.0K 次观看.  University of Pavia. 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.

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

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

科研

遗传学

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

发育生物学

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

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 ...

环境科学

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

注射格里鲁斯双形蛋

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

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 ...

生物学

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

生物工程

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

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, ...

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

摘要

探索更多视频

激光辅助畜禽受精卵细胞质显微注射

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

胚胎显微注射和电的脊索动物

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

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

注射格里鲁斯双形蛋

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

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎

用于显微注射的方法

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

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

摘要

探索更多视频

激光辅助畜禽受精卵细胞质显微注射

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

胚胎显微注射和电的脊索动物

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

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

注射格里鲁斯双形蛋

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

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

用于显微注射的方法

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

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎