Figure 2

The structure and function of transposon LA513. LA513 is a non-autonomous piggyBac-based transposon of 8.4 kb. Transgenics are readily identified by red fluorescence due to expression of DsRed2. tTAV is a tetracycline-repressible transcriptional activator [28, 48]. Here, tTAV is under the control of its own binding site, tetO, a minimal promoter from Drosophila hsp70, and a 3' UTR sequence from Drosophila fs(1)K10 [49]. In the absence of tetracycline, tTAV binds to tetO and drives expression of more tTAV, in a positive feedback loop. In the presence of tetracycline, tTAV binds tetracycline; this tetracycline-bound form does not bind tetO and so does not lead to expression of more tTAV. Consequently, this construct gives very high levels of expression of tTAV in the absence of tetracycline, but only low, basal expression in the presence of tetracycline. High level expression of tTAV is toxic, possibly due to the interaction of the VP16 domain with key transcription factors, so this construct provides a tetracycline-repressible lethal system [28]. Construct LA882 is very similar to LA513; the principal difference is the use of the IE-2 promoter from the baculovirus OpNPV to drive expression of the DsRed2 marker, in place of Act5C.