PMC

Phenotype of line V_B, eIR-19₁ and eIR-A₃. EGFP-expressing A.stephensi larvae from transgenic line V_B (left) and F₁ larvae derived from the cross between line V_B and eIR-A₃ (center) or eIR-19₁ (right). In the center and right panels, DsRed fields are superimposed upon EGFP fields to show co-localization (yellow). Line eIR-A₂ exhibited a phenotype similar to that of line eIR-A₃ (data not shown). Larvae were photographed at 10× magnification 5 days post-hatching.

Plasmid pIR-EGFP and Southern blot analysis of transgenic lines. (a) Schematic representation of transformation vector pIR-EGFP. Actin5C, D.melanogaster Actin5C promoter; HspT, D.melanogaster Hsp70 terminator sequence; ML, minos left arm; MR, minos right arm; IR-EGFP, EGFP inverted repeat; DsRed, DsRed selectable marker. The restriction sites (H, HincII; E, EcoRI; N, NotI) and the probes (M, EGFP-S and HST, represented by black bars) used in the Southern blot analyses are indicated. A 1 kb scale marker is shown. (b) Southern blot analyses of genomic DNA from 17 transgenic lines digested with HincII and hybridized with probe M. (c) Southern blot analyses of genomic DNA from selected lines eIR-A₁, eIR-A₂, eIR-A₃, eIR-B₁, eIR-C₂ and eIR-19₁, digested with EcoRI/NotI and hybridized with probe EGFP-S (upper) or probe HST (lower). The size marker is in kilobases (kb). A₂, eIR-A₂; A₃, eIR-A₃; B₁, eIR-B₁; 19₁, e-IR-19₁; A₁, eIR-A₁; C₂, eIR-C₂.

Quantification of RNAi in A.stephensi larvae. (a) The intensity of EGFP protein expression in the F₁ and F_{1 Backcross} progeny of the crosses between the eIR lines and line V_B was calculated using the Lucia G image processing and analysis software. Gray bars indicate normalized EGFP expression levels in F₁ progeny (one transgene copy); black bars indicate normalized EGFP expression in F_{1 Backcross} progeny (two transgene copies). (b) Relative EGFP mRNA levels in F_{1 Backcross} progeny, as determined by real-time PCR. Black bars indicate normalized EGFP mRNA levels. EGFP protein and RNA expression levels were normalized to control larvae derived from a cross between line V_B and wild-type mosquitoes (V_B) (white bars). The plotted data show mean values ± SE. RED1, MinRED1; A 2, eIR-A₂; A 3, eIR-A₃; B 1, eIR-B₁; 19 1, e-IR-19₁; A 1, eIR-A₁; C 2, eIR-C₂. Asterisks indicate the significance value of the result. *Significant, P < 0.05; **highly significant, P < 0.01.