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Figure 8


Fig. 7. Site-specific integration in Drosophila. (A) {Phi}C31 integrase-mediated transgenesis using single attP docking sites. Docking sites are transposons, such as P elements (Groth et al., 2004), piggyBac (Venken et al., 2006) or Mariner (Bischof et al., 2007), that contain a single attP recombination site and a marker 1, and that are integrated into the genome. A plasmid containing an insert, marker 2 and an attB recombination site, can then integrate into the docking site when {Phi}C31 integrase is provided. Correct recombination events between attP and attB are identified using marker 2. They result in two hybrid sites, attL and attR, that are no longer a substrate for {Phi}C31 integrase - the reaction is therefore irreversible. (B) Cre- and FLP-mediated RMCE. Docking site transposons (with 5' and 3' transposon termini), such as P (Oberstein et al., 2005) or piggyBac (Horn and Handler, 2005) elements, contain marker 1 flanked by heterotypic direct-oriented recombination sites (RS) `RS1' (loxP or FRT, gray) and `RS2' (such as lox2272 or F3, purple). The RMCE plasmid, containing marker 2 flanked by a similar configuration of heterotypic recombination sites, can integrate when Cre or FLP is provided. Correct recombination events are identified by the absence of marker 1 and presence of marker 2. Recombination can be partial (single integration events are not shown) and is reversible. (C) {Phi}C31 integrase-mediated RMCE. A docking site P element transposon (5'P and 3'P element termini) (Bateman et al., 2006) contains a marker 1 flanked by inverted attP recombination sites. The RMCE plasmid, containing insert flanked by inverted attB recombination sites, can integrate when {Phi}C31 integrase is provided. Correct recombination events, between both attP and attB sites, are identified through absence of marker 1 and result in hybrid sites, attL and attR, that are no longer substrates for {Phi}C31 integrase. The integrated DNA can be in either orientation (arrows).





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