In order to investigate the feasibility of establishing a stable and heritable RNAi technology in mosquitoes, EGFP from a transgenic A.stephensi line was selected as the target gene. It was reasoned that EGFP could provide an impartial model to determine the feasibility of stable RNAi in Anopheles. The rationale for this was based on the consideration that silencing of the EGFP transgene would produce a neutral phenotype for the mosquito, avoiding the risks of selection bias associated with high silencing activity against important or vital endogenous genes.
The RNAi gene was designed to produce intron-spliced dsRNA, which our previous studies had demonstrated could mediate transient RNAi with high efficiency against EGFP
). In that study, EGFP
silencing achieved in transient transfection experiments of Anopheles
cells and larvae averaged 93 and 69%, respectively. When stably expressed, this RNAi gene suppressed EGFP
expression by up to 73% at the protein level and 78% at the RNA level. The intron-containing RNAi gene was utilized here on the assumption that it would be less susceptible to the genomic rearrangements reported to occur in constructs containing uninterrupted repeats (23
). Our analysis, however, revealed the complete absence of the RNAi unit in two of the transgenic lines studied. This finding could be explained by recombination events that took place either within the plasmid itself or genomically. Recombination may have been triggered by the presence of the symmetrically parallel RNAi and DsRed
transcription units (Fig. a), sharing identical promoter and terminator sequences, rather than as a direct consequence of the presence of the IR in the RNAi gene itself. Importantly, the integrated constructs showed genomic stability beyond nine generations, which argues for construct instability at the time of injection.
Our data clearly indicate that silencing effects were improved by doubling the RNAi gene copy number, in accordance with similar studies using IR transgenes in Drosophila
). Levels of EGFP protein expression were approximately halved when a second copy of the IR transgene was provided in two of the crosses. Furthermore, silencing efficiency appeared to be dependent upon the site of integration of the RNAi gene. Silencing was only observed in those lines where the expression pattern of the EGFP
target gene and the RNAi gene, as determined by DsRed
expression, overlapped. This is in agreement with studies of transgene-mediated RNAi in Drosophila
), indicating that RNAi is not systemic and does not spread across mosquito cells. Whether this suggests a lack of dsRNA amplification in Diptera, common to Neurospora
), plants (26
) and C.elegans
), is yet to be established, but it may simply reflect the postulated inefficiency of dsRNA formation from transgenes, possibly because nuclear RNA-binding proteins prevent dsRNA annealing (28
While it is impossible to extrapolate the results of this study to endogenous mosquito genes, the level of gene silencing achieved here is encouraging despite the fact that we were unable to completely abolish the EGFP phenotype. Since RNAi only knocks down target gene expression, it could offer an advantage in performing functional genomic studies on vital genes and produce a range of useful phenotypes that are missed in classical knockout studies. Because minos reliably creates multiple genetic lines, an array of useful hypomorph phenotypes is likely to be generated for most studies.
The results reported here represent the first example of stable RNAi in Anopheles
. This approach can now be applied to study mosquito molecules that have been postulated to function as receptors for malaria parasite development. Furthermore, stable RNAi could greatly contribute to the understanding of important mosquito physiological functions, including chemoreception, locomotion, osmoregulation and homeostasis, taking advantage of the recently published A.gambiae
genome sequence (16