A transgenic sensor strain for monitoring the RNAi pathway in the yellow fever mosquito, Aedes aegypti

Abstract

The RNA interference pathway functions as an antiviral defense in invertebrates. In order to generate a phenotypic marker which "senses" the status of the RNAi pathway in Aedes aegypti, transgenic strains were developed to express EGFP and DsRED marker genes in the eye, as well as double-stranded RNA homologous to a portion of the EGFP gene. Transgenic "sensor" mosquitoes exhibited robust eye-specific DsRED expression with little EGFP, indicating RNAi-based silencing. Cloning and high-throughput sequencing of small RNAs confirmed that the inverted-repeat transgene was successfully processed into short-interfering RNAs by the mosquito RNAi pathway. When the A. aegypti homologues of the genes DCR-2 or AGO-2 were knocked down, a clear increase in EGFP fluorescence was observed in the mosquito eyes. Knockdown of DCR-2 was also associated with an increase in EGFP mRNA levels, as determined by Northern blot and real-time PCR. Knockdown of AGO-3, a gene involved in the germline-specific piRNA pathway, did not restore EGFP expression at either the mRNA or protein level. This transgenic sensor strain can now be used to identify other components of the mosquito RNAi pathway and has the potential to be used in the identification of arboviral suppressors of RNAi.

Figure 1

Transformation of Ae. aegypti with the pMos3xP3-sensor construct. (A) Schematic representation of a hypothetical insertion. Block arrows indicate the left (LH) and right (RH) arms of the Mos1 transposon, dotted line indicates mosquito chromosomal DNA. Small arrowheads indicate the approximate start of transcription sites for each 3xP3 promoter. Restriction enzyme recognition sites for EcoR I (R1), Sac I (S1) and Pst I (P) are indicated. Portions of the 3xP3-sensor construct recognized by the probe during Southern analysis are indicated with solid bars. (B) Southern analysis of genomic DNA isolated from lines #2, 9.1 and 9.6 or host kh^w, digested with restriction enzymes EcoR I, Sac I or Pst I. Approximate molecular size markers are shown at left (in kbps). Arrowhead indicates the expected size of common Pst I-generated hybridization fragment.

Figure 2

Identification of EGFP siRNAs. (A) Short-interfering RNAs cloned from 3xP3-sensor heads (line #2) correspond to the EGFP inverted repeat sequence. Each base in the 720 nucleotides of the EGFP ORF was assigned a score based on the number of times it appeared in an siRNA. Separate scores were calculated for each strand, with siRNAs corresponding to the sense (s) strand plotted in the positive direction and those corresponding to the antisense (as) strand plotted in the negative direction. The region corresponding to the 505 bp inverted repeat (EGFPir) is noted. (B) Length of EGFP siRNA sequence as determined by blastn of siRNA library following removal of linker sequences, as described in Materials and Methods.

Figure 3

Transgenic 3xP3-sensor mosquitoes express EGFP when RNAi is compromised. (A) Schematic representation of the conditional expression of EGFP in 3xP3-sensor mosquitoes. (B) Two representative 3xP3-sensor mosquitoes (line #2) photographed under white light (left), DsRED fluorescence (middle) and EGFP fluorescence (right) following knock-down of either AaAGO-3 or AaDCR-2.

Figure 4

Recombinant dsSINVs induce effective gene knock-down. Real-time PCR was performed on cDNA derived from 3xP3-sensor transgenic mosquitoes. Levels of AaDCR-2 and AaAGO-3 transcript were normalized with AaElav, an endogenous gene expressed in mosquito head tissue (Adelman, unpublished). The first two bars represent relative levels of AaDCR-2 transcript in uninjected and dsSINV-AaDCR-2-injected 3xP3-sensor mosquito heads, while the second two bars represent relative levels of AaAGO-3 transcript in uninjected and dsSINV-AaAGO-3-injected 3xP3-sensor mosquito heads.

Figure 5

Qualitative analysis of 3xP3-sensor mosquitoes following knock-down of putative RNAi genes. (A) 3xP3-sensor mosquitoes (line #2) were scored based on intensity of EGFP fluorescence. The percent of total mosquitoes scored at each value (1–4) is plotted, with the average for each sample and the total number of mosquitoes scored (n) given in parentheses. Results represent the sum of three replicate experiments performed during the G₄-G₈ generations for uninjected, dsSINV-DCR-2 and dsSINV-AGO-3 and two replicates for dsSINV-AGO-2. Sample photographs below the graph illustrate the approximate scoring guide. (B) Northern analysis of 3xP3-sensor mosquitoes following knock-down of putative RNAi genes. RNA from uninjected, dsSINV-AaDCR-2, or dsSINV-AaAGO-3 injected heads was probed for EGFP mRNA. Gel photograph of rRNA prior to transfer serves as loading control. (C) Real-time RT-PCR (two-step) to detect EGFP mRNA expression in 3xP3-sensor #2 heads following knockdown of AaDCR-2 or AaAGO-3. Samples were compared to EGFP expression in uninjected 3xP3-sensor #2 mosquito heads (−).