埃及斑蚊胚胎显微注射获得转基因蚊子

Summary

在这个视频，Nijole Jasinskiene演示方法生成的转基因埃及伊蚊蚊子，登革热的病媒。正确准备显微注射针，dessicating胚胎，并执行显微注射​​技术演示。

Abstract

在这个视频，Nijole Jasinskiene演示方法生成的转基因埃及伊蚊蚊子，登革热的病媒。正确准备显微注射针，dessicating胚胎，并执行显微注射​​技术演示。

Protocol

在显微注射前的制备血喂蚊子：从周一至在上周四周五饲料女性注射。 准备使用方案3或2（见材料）的玻璃针。 铺设管的胚胎，是果蝇文化小瓶。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，它在盖玻片边缘结束。 准备干燥油（见材料） 150毫升塑料烧杯中，用湿药棉覆盖的湿滤纸（80毫米滤?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
1x Injection solution	Buffer			5mM KCl, 0.1mM sodium phosphate, ph 6. 8
Construct plasmid	DNA			0.5 mg/ml.
Helper plasmid	DNA			0.3 mg/ml
EndoFree Plasmid kit	Kit	Qiagen
Millex-GV column			SLGV RO4 NL	To clean DNA.
Glass puller	Instrument	Sutter Instrument Co	Model P-87	Heat-815, Pull-10, Vel-10, Time-100. Heat-700, Pull-30, Vel-36, Time-50
Quartz puller	Instrument	Sutter Instrument Co	Model P-2000	Heat-275, Fil-3, Vel-38, Del-250, Pull-141
Injection glass needles				Prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 um micropipette puller. When the needle is new, the tip is sealed and usually breaks in the first or second injection or the glass needle tip can be beveled using a Brown type micro-pipette beveller (Sutter Instrument).
Aedes aegypti	Animal			Male and female mosquitos, to obtain embryos.
Drosophila culture vial				For mosquito egg-laying.
Halocarbon oil	Reagent			Oil for desiccation: Halocarbon oil 700:Halocarbon oil 27(1:1)
Forceps No5	Tool
Fine paintbrush		Sable	No. 0000
Whatman paper	Tool
Microloader	Tool	Eppendorf	5242 956.003
Transjector	Tool	Eppendorf
Microscope		Leica		With moving stage and micromanipulator.
3MM Whatman paper
double sided tape