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Fig. 1. Schematic representation of DNA constructs, and molecular strategy of Cre/lox-mediated gene deletion and re-integration. Donor locus developed by cobombardment of plasmids, p35Shpt and pVS55, contains loxP- and lox75-flanked neomycin phosphotransferase gene (npt) (promoterless) and ß-glucuronidase gene (gusA). Target locus developed by the integration of pVS52 construct contains a cre transcription unit and a target lox76 site. Cre/lox recombination is expected to excise lox-flanked DNA fragments in the form of extra-chromosomal circles containing lox75 that may interact with the genomic lox76 site in the target T5 locus to generate a predictable integration locus. Lox sites are represented as arrowheads between filled (mutant) or empty (wild-type) bars. EcoRI (E) sites are shown for each vector and expected fragment sizes are given in kb. Position of PCR primers are indicated by arrowheads. DNA fragments used to probe Southern blot are underlined. 35S, CaMV 35S promoter; hpt, hygromycin phosphotransferase gene; nos, nopaline synthase transcription termination signal; pubi, maize ubi-1 promoter; loxP wild-type lox site; lox75, mutant (left arm) lox site; lox76, mutant (right arm) lox site; dmlox, double mutant lox site.
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