Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

Summary

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Abstract

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Protocol

Preparation in advance of microinjection: Blood feed mosquitoes: for injection from Monday to Wednesday feed females on the previous Friday. To inject Thursday and Friday, feed females on Monday of the same week. Prepare the Quartz needles using program 2 and isotonic buffer (see Materials). “Laying tube” for the embryos is the regular Drosophila culture vial. Wet cotton wool in the bottom, with a wet disk of filter paper covering it. Prepare plast…

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