Transformation construct and Southern blot hybridization. (A) Schematic representation of the transformation vector pBac(3xP3RED)AgApy used for the An. gambiae germ line transformation. The piggyBac left (pBacL) and right (pBacR) arms, the 3xP3 promoter, the DsRed transformation marker, the SV40 terminator, the AgApy promoter, the LacZ reporter gene and the bgh terminator are shown. Bars represent the hybridization probes: probe B, hybridizing to the left and right inverted terminal repeats, and probe P, corresponding to a fragment of the AgApy promoter. E and H indicate Eco RI and Hind I restriction sites. (B and C) Hind III digested genomic DNA from the different An. gambiae transgenic lines are indicated on the top. (B) Hybridization with probe B (Fig. 1A), which detects the piggyBac arms: each insertion is expected to yield two bands of variable size. (C) Hybridization with probe P (Fig. 1A), which detects the AgApy promoter: here, each insertion is expected to yield four bands of fixed size irrespective of transgene copy number, two from the endogenous AgApy gene (end) and two from the transgene (tra). The numbers on the left refer to the molecular weight marker (Kbp).