Stable and heritable gene silencing in the malaria vector Anopheles stephensi

Abstract

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles . This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Innate immunity in the malaria vectorAnopheles gambiae:comparative and functional genomics

“…To date, only a few promoters have been characterized and used to drive gene expression in mosquitoes. These include Aedes vitellogenin (Shin et al, 2003a), Drosophila actin5C (Brown et al, 2003) and Anopheles carboxypeptidase promoter . The characterization of mosquito promoter sequences could be greatly aided by utilization of the Cre-LoxP system, which was shown recently to be active in Ae.…”

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi

“…An. stephensi is amenable to genetic manipulations such as transposon-based germline transformation [10], genome-wide mutagenesis [11], site-specific integration [12], genome-editing [13], and RNAi-based functional genomics analysis [14]. Our understanding of the interactions between An.…”

Transgenesis, Paratransgenesis and Transmission Blocking Vaccines to Prevent Insect-Borne Diseases

“…Stable transformation of mosquitoes with two inverted repeats of the same gene to induce assembly of double-strand RNAs (dsRNAs) and activation of the RNAi pathway has also been obtained (Brown et al 2003;Bian et al 2005). This strategy takes advantage of the RNAi mechanism to block expression of insect molecules associated with vectorial competence (Bian et al 2005), or it can directly target viral replication within insect tissues (Franz et al 2006).…”

“…stephensi expressing sense and anti-sense RNAs targeting EGFP (Brown et al 2003). EGFP dsRNA-expressing mosquitoes were crossed with a transgenic mosquito line that expresses EGFP.…”

“…EGFP dsRNA-expressing mosquitoes were crossed with a transgenic mosquito line that expresses EGFP. The double transgenic offspring displayed lower level of EGFP expression than the parental line expressing it, indicating the effect of the RNAi machinery reducing the expression of the EGFP transgene (Brown et al 2003). Ae.…”