Fig. 3

φC31-mediated site-specific integration and RMCE. (A) Scheme for site-specific germline transformation. D. suzukii line 06_F5M2 [52] carries construct HMMA006 that contains an attP recombination target sequence, which - in the presence of a helper plasmid providing φC31 integrase (HMMA098) - is targeted by construct HMMA182 carrying the corresponding attB recombination site to integrate the complete plasmid. The integration leads to a modification of the transgenic insert, which can be used for additional integration of transgenes (light green “?”) as well as transgene stabilization by removing part of the transgenic composition by piggyBac excision [39]. (A′-A‴) Integration can be detected by the addition of the EGFP marker. (B) RMCE to generate diverse transgenes at the same genomic position. D. suzukii line carrying construct HMMA185 is targeted by construct HMMA336 in the presence of a helper plasmid (HMMA098) providing φC31 integrase to exchange marker genes and integrate a specific cargo gene (TRE-Cas9). (B′-B‴) RMCE can be detected by the replacement of the DsRed marker with the EGFP marker. Images of a male fly of each indicated line are taken with cold light (A′,B′), RFP filter (A″,B″), or EYFP filter (A‴,B‴)