注射的。 stephensi胚胎生成抗药性疟疾的蚊子
Summary
按蚊的蚊子是疟疾居住在印度和整个亚洲的载体。本视频演示了执行这个品种与转基因，这将赋予抵抗疟疾的蚊子microinjections技术。在此视频中演示的方法大多是适用于其他蚊种的显微注射技术。
Abstract
引入外源基因的蚊子的基因组需要针对特定物种的利益显微注射技术。这个视频协议演示由詹姆斯实验室用于microinject到按蚊改造的蚊子的产生胚胎的DNA结构的方法。都说明，为准备显微注射针，收集和准备胚胎和执行显微注射技术。
Protocol
在显微注射前准备： 血喂蚊子：从周一至周三上周五饲料女性注射。要在同一个星期的周四和周五，饲料女性在周一注入。 准备使用方案2和等渗缓冲液（见材料）的石英针。 “铺设管”的胚胎是普通果蝇文化小瓶。湿脱脂棉在底部，一个覆盖它的过滤纸的湿磁盘。 准备贴双面胶的一端塑料盖滑。修剪磁带覆盖滑，因此，它在盖玻片边缘结…
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Injection Quartz needles | Sutter Instrument | O.D.- 1.0 mm, I.D.-0.70, 10 cm length, are prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 mm micropipette puller. | ||
| Quartz puller | Tool | Sutter Instrument Co | P-2000 | Set-up: Heat-275, Fil-3, Vel-38, Del-250, Pull-141 |
| EndoFree plasmid kit | Quiagen | |||
| Isopropanol | ||||
| Millex-GV column | SLGV RO4 NL | for DNA cleaning | ||
| Construct DNA | 0.5 mg/ml | |||
| Helper plasmid | 0.3 mg/ml | |||
| Injection solution | Buffer | 1x solution: 5mM KCl, 0.1mM sodium phosphate, ph 6. 8 | ||
| Isotonic buffer | see recipe in the protocol section | |||
| Dessication oil | Halocarbon oil 700:Halocarbon oil 27(1:1). Mix well. Prepare in advance of microinjection. | |||
| Blood | to feed mosquitos | |||
| Anopheles stephensi | Animal | Mosquitos, male and female. | ||
| Drosophila culture vial | for egg laying | |||
| Petri dishes | with isotonic buffer, for embryo hatching | |||
| Fine paintbrush | Sable # 0000 | |||
| 3MM Whatman paper | ||||
| Forceps #5 | ||||
| double sided tape | ||||
| Microloader | Eppendorf | 5242 956.003 | ||
| Transjector | Eppendorf | |||
| Microscope | Leica | with moving stage and micromanipulator, set at 10x magnification |
Cite This Article
Terenius, O., Juhn, J., James, A. A. Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes. J. Vis. Exp. (5), e216, doi:10.3791/216 (2007).