High efficiency germ-line transformation of mosquitoes

Abstract

The ability to manipulate the mosquito genome through germ-line transformation provides us with a powerful tool for investigating gene structure and function. It is also a valuable method for the development of novel approaches to combating the spread of mosquito-vectored diseases. To date, germ-line transformation has been demonstrated in several mosquito species. Transgenes are introduced into pre-blastocyst mosquito embryos using microinjection techniques that take a few hours, and progeny are screened for the presence of a marker gene. The microinjection protocol presented here can be applied to most mosquitoes and contains several improvements over other published methods that increase the survival of injected embryos and, therefore, the number of transformants. Transgenic lines can be established in approximately 1 month using this technique.

Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

“…gambiae and more technically challenging 22,23 . Since then, several modifications to the protocol have improved the efficiency considerably, as outlined in Lobo et al 24 . Technical details for producing transposon-mediated transgenic mosquitoes including An.…”

“…Technical details for producing transposon-mediated transgenic mosquitoes including An. gambiae have been published by several groups [24][25][26][27] .…”

“…At that stage they are suitable for alignment and injection 24,25 and are transferred to the injection room conditioned at 18-20°C to slow down embryo development.…”

“…Check also the DNA purity by measuring OD 260/280, which should not be less than 1.8. Embryo alignment for microinjection TIMING 10-20min/ batch of 50 embryos CRITICAL: Methods for aligning Anopheles eggs have been well described [24][25][26][27]46 . Our procedure is very similar to those described in 24,26 .…”

“…We select embryos on several criteria as described by Lobo et al 24 to maximize survival after injection. White or yellowish eggs, eggs that are too dark, small or thin eggs, and those with visible defects in the chorion or in their morphology, are discarded.…”