In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

Abstract

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.

Role of BmDredd during Apoptosis of Silk Gland in Silkworm, Bombyx mori

“…The protocol is easy and versatile that it has been adapted to many other species including mice, rat, chicken and mosquito [30–33]. We transiently overexpressed the BmDredd in vivo in silkworm SG using TurboFect in vivo transfection Reagent (Fig 4A and 4B).…”

RNA Interference for Mosquito and Mosquito-Borne Disease Control

“…In this case, eggs were incubated in dsRNA until the time of hatching resulting in 56%–90% reduction in target gene expression in a dose-dependent manner [118]. Knockdown in the embryo also has been demonstrated indirectly through plasmid expression vectors [119,120]. Here plasmid injected into adults passed to the progeny and induced up to 99% suppression of the target gene following heat shock in progeny.…”

“…In larvae, RNAi is functional throughout the course of development with knockdown being inducible either by direct exposure or via inheritance from exposed parents [119,120,122,123]. Direct exposure of larvae has proven effective when administering RNAi triggers through injection, per os , soaking, or rehydration in aqueous solutions [12,13,75,123,124,125].…”

“…Pupal development can be disrupted following direct injection of RNAi triggers for prophenoloxidase III [19,85]. Although pupae cannot be exposed per os, RNAi trigger exposure in larvae, or even adult female F0s could provide sufficient long term persistence to suppress pupal genes [119,120]. Indeed Mysore et al (2014) suppressed pupal brain development following larval exposure to dsRNA chitosan nanoparticles targeting the single-minded gene (AAEL011013) [128].…”

“…Bacterial production of dsRNA still requires the dsRNA to be delivered to the mosquito following production, which has proven effective per os [13]. Alternatively, plasmids with insect specific promoters such as β-tubulin or actin 5C can facilitate production of the dsRNA within target cells of the mosquito [17,119,120,153,154,155,156]. Insect expression plasmids can also persist through multiple generations, with dsRNA production being inducible when using heat shock promoters [119,120].…”