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.
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Method Article
Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes
In This Article
Summary
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 plastic cover slip by sticking double-side tape to one end. Trim tape to cover slip so that it ends at edge of cover slip.
- Prepare oil....
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Materials
| Name | Company | Catalog Number | Comments | |
|---|---|---|---|---|
| Injection Quartz needles | Sutter Instrument Co. | 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 | Qiagen | |||
| 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 Microsystems | with moving stage and micromanipulator, set at 10x magnification |
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