Mosquito Embryo Microinjection under Halocarbon Oil or in Aqueous Solution

Abstract

The process of genetically modifying mosquitoes requires skilled delivery of reagents for modification. Plasmids, RNA, DNA, and/or protein must be transported into the developing embryo during an appropriate time in development when these agents will have access to the genome. Embryo microinjection has been the main method by which such modifying agents have been delivered. Ideally the microinjection process will deliver these modifying agents in sufficient quantity to effect the genetic modification without severely damaging or killing the injected embryo in the process. As semiaquatic insects, mosquitoes have embryos that are susceptible to desiccation and the degree to which embryos are susceptible is based on species. Two microinjection methods are outlined here. The first method describes embryo microinjections performed under Halocarbon-27 oil. The oil is used to reduce desiccation during the injection process. A second method limits desiccation by injecting the mosquito embryos in water. In both procedures, the embryos are first aligned and then injected before the embryos cellularize, ∼1 h and 45 min after oviposition.