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Comparative analysis of DNA vectors at mediating RNAi in Anopheles mosquito cells and larvae

Anthony E. Brown, Andrea Crisanti and Flaminia Catteruccia^*

Department of Biological Sciences, SAF Building, Imperial College London, Imperial College Road, London SW7 2AZ, UK

View larger version (24K): [in a new window]   Fig. 1. Plasmid maps and predicted structure of dsRNA transcripts. (A) Schematic representation of target plasmid pMinEGFP and control plasmid pMiRED. (B) Design of EGFP inverted repeats (EGFP-IR) and predicted structure of dsRNA transcript. Filled arrows pointing in opposite directions represent the EGFP-IR. Spacer regions are shown. (C) Schematic representation of generic dsRNA-expression plasmid, pIR-EGFP. Each IR-EGFP construct was cloned into pMiRED within the arms of minos. Actin5C, D. melanogaster actin5C promoter; HspT, D. melanogaster Hsp70 terminator sequence. The EGFP-, EGFP-IR- and DsRed-encoding genes are indicated by filled arrows. ML, minos left arm; MR, minos right arm.

View larger version (30K): [in a new window]   Fig. 2. Fluorescence micro-photographs of A. gambiae Sua 4.0 cells and A. stephensi larvae. In A–D, column 1 shows DsRed fluorescence, column 2 shows EGFP fluorescence and column 3 shows DsRed fields superimposed upon EGFP fields to show co-localisation (yellow). (A) Sua 4.0 cells transfected with pMinEGFP and pMiRED show perfect co-localisation. (B) Sua 4.0 cells transfected with pMinEGFP and pIR-EGFP465-Linker show virtually no EGFP expression. (C) Wild-type A. stephensi larvae injected with pMinEGFP and pMiRED show exact co-localisation. (D) Wild-type A. stephensi larvae injected with pMinEGFP and pIR-EGFP465-Linker show very limited EGFP expression. Cells were captured on a Nikon inverted microscope at 20x magnification 48 h post-transfection. Larvae were photographed at 10x magnification 48 h post-hatching. (E) Four-day-old EGFP-expressing A. stephensi larvae from transgenic line VB injected with pMiRED (column 1) or pIR-EGFP465-Linker (column 2). Column 3 shows localisation of DsRed expression from pIR-EGFP465-Linker in the same larva.

View larger version (19K): [in a new window]   Fig. 3. Quantification of RNA interference in A. gambiae Sua 4.0 cells and A. stephensi larvae. (A) A. gambiae Sua 4.0 cells co-transfected with pMinEGFP and one of the dsRNA-expressing plasmids. (B) Wild-type A. stephensi larvae injected with pMinEGFP and one of the dsRNA-expressing plasmids. The intensity of EGFP and DsRed expression was calculated using the Lucia G image-processing and analysis software. Red and green fluorescence levels were normalised to control experiments in which pMinEGFP and pMiRED were delivered. Black bars indicate normalized EGFP expression levels; grey bars indicate normalized DsRed expression. The plotted data show mean values ± S.E.M. from >20 fields captured in three separate transfection experiments or >20 injected larvae per experiment. All reductions in EGFP expression were found to be highly significant (P<0.01). DsRed expression levels did not vary significantly between individual experiments (P>0.05). S 465, pIR-EGFP465S; NS 465, pIR-EGFP465NS; Linker 465, pIR-EGFP465-Linker; S720, pIR-EGFP720S. (C) EGFP-silencing after injection of either pMiRED (broken line) or pIR-EGFP465-Linker (solid line) into stable EGFP-expressing transgenic line VB. The intensity of EGFP expression was calculated 3, 4 and 5 days post-hatching.