Fig. 2

Site-specific integration into T5 locus. The development of T5 line is described earlier [28]. a T5 site contains lox76 (right arm mutant) between maize ubiquitin-1 (Ubi) promoter and cre coding sequence, and a hygromycin selection marker (HPT); b Donor plasmid pNS27 (pUC backbone), designed for site-specific integration into T5 site, contains between loxP (wild-type) and lox75 (left arm mutant), a promoter-less Bar gene (*), GFP gene (35S:GFP), promoter-less NPT gene, and FRT site. Bar gene is flanked by I-SceI target sites, and a single CCR5 site is present downstream of NPT gene. Each gene contains nos 3′ transcriptional termination (nosT) sequence (not shown); c Introduction of pNS27 into T5 cells, that already express Cre activity, results in the integration of the donor construct into T5 site via lox75 x lox76 recombination, generating precise site-specific integration (SSI) that contains loxP downstream of Ubi, and double-mutant lox78 upstream of cre (black triangles); d PCR analysis of T5-SSI lines (S1 – S5) using primers shown above; d Southern hybridization of EcoR1 (e) digested genomic DNA of SSI lines with GFP and nosT fragments. M, 1-kb DNA ladder. The expected PCR and EcoRI fragments from precise SSI site are shown in (c)