RNAi介导的基因敲除和<em>在体内</em在成年女性利尿含量<em>埃及伊蚊</em>蚊子
Summary
在这个协议中，我们结合RNAi介导的基因沉默<em>在体内</em>利尿法研究感兴趣的基因的影响击倒蚊虫液的排泄。
Abstract
这个视频协议演示了一个有效的技术击倒在昆虫特定基因，并进行一种新的生物活性来衡量排泄率。这种方法可以用于获取更好地了解昆虫的利尿过程，是在利尿吸血节肢动物，能够占用大量的液体，在一个单一的血粉的研究，特别是有用的。
这RNAi介导的基因敲除结合在体内利尿法是由汉森实验室研究RNAi介导水通道蛋白基因埃及伊蚊利尿1击倒的影响。
该协议是在设置两部分：第一个示范说明如何构建一个简单的蚊子喷射装置及如何准备和注入的蚊子胸部RNAi介导的基因敲除的dsRNA。第二个示范说明如何确定在使用延命蚊子的排泄率。
Protocol
第一部分- RNAi介导的基因敲除成年埃及伊蚊蚊。实验概述， 见图1。 1。双链RNA的合成对利益和控制的dsRNA基因合成的双链RNA。注：我们建议发展中国家引物的PCR片段范围在300至500个碱基对位于特定基因2月底3'和T7引物序列在5'端（5'-TAA TAC GAC中TCA的CTA重视。豪GG-3'）。片段的唯一性，应确认BLASTN程序分析3。 使用的Ambio…
Discussion
RNAi的协议，用于已开发在Raikhel亚历山大实验室在加州河滨大学6,7和类似由加弗和Dimopoulos 4发表了一份协议。这个视频协议中的实验方法可以用来研究有关的基因在昆虫体内设置利尿。作为一个简单的“利尿模型系统，吸引昆虫，马氏管，排泄器官几代科研人员的利益。这个器官是涉及通关8和果蝇的工作已确定在马氏管生理9,10，现在可以在其他昆虫…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢她的这个协议的批评维多利亚木匠。
Materials
| Name of reagent or equipment | Company | Catalogue number | Comments |
| MEGAscript T7 High Yield Kit | Ambion, Inc. | AM1334 | |
| PBS buffer | Sigma-Aldrich | P4417 | |
| Plastic tubing | Local vendor | PVC | |
| 1 ml plastic pipette tip | VWR | 83007-376 | Blue tip |
| 1 ml syringe | Becton, Dickinson and Company | 309602 | |
| Scissors | Local vendor | ||
| Metal needle | Carolina Biologicals | 654307 | Size 5 |
| Fly pad | Genesee Scientific | 789060 | |
| Battery-powered aspirator w/ collection vial | UPMA Labs | IPMM 2000 | |
| Fine tip forceps | World Precision Instruments | 14095 | |
| Glass capillary needles | World Precision Instruments | 1B200-6 | |
| Stereo dissection microscope | Leica Microsystems | S6D | |
| Analytical precision balance | Mettler Toledo | AB54S | |
| Sucrose | Sigma-Aldrich | 84097 | |
| One pint waxed lined cardboard cups | Local vendor | Manufactured soup cups | |
| Mesh net | Local vendor | plastic fly gauze |
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Cite This Article
Drake, L. L., Price, D. P., Aguirre, S. E., Hansen, I. A. RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes. J. Vis. Exp. (65), e3479, doi:10.3791/3479 (2012).