Fig. 4

Site-specific integration into T5-SSI by FLP-FRT recombination. a Structure of the T5-SSI obtained through Cre-mediated site-specific integration (see Fig. 2), note the presence of promoter-less (*) NPT gene and FRT site for the next round of site-specific integration; b Donor vector, pNS35, contains FRT flanked maize ubiquitin promoter (Ubi) oriented to trap NPT gene in the marker-free SSI site, and the gus gene as gene-of-interest; c Delivery of pNS35 and FLPe gene generates SSI locus through FRT x FRT recombination. This SSI is selectable due to the activation of NPT gene; d PCR analysis of geneticin resistant SSI lines showing the presence of the expected 1.4 kb SSI footprint; e Southern hybridization of SSI lines (A – E) on HindIII-digested genomic DNA probed with Ubi fragment. HindIII (H) sites, fragment sizes, primer sites are shown in the SSI structure (c). DNA sizes are shown in kb