Figure 1.

Strategy to obtain Cre-mediated cassette exchange. A, Chromosomally introduced target cassette (T-DNA pSDM3164). The bar gene is flanked by the loxP site (large black arrow) and the lox5171 site (large dashed arrow) in inverted orientation, with the strong CaMV 35S promoter and double enhancer sequence (DE35S) adjacent to the cassette providing resistance to PPT. B, Exchange T-DNA pSDM3327 contains the nptII coding sequence and gus expression unit driven by the BigMac promoter, flanked by lox sites in similar configuration as the bar cassette. The nos transcription terminator, tnos, and three translation stop signals in the different reading frames are included to prevent nptII expression from neighboring sequences upon random integration. The conditional negative selectable marker codA with the 34S promoter is placed outside the cassette. Arrow 1 indicates direct cassette exchange via a double crossover reaction loxP × loxP and lox5171 × lox5171, mediated by Cre that is provided via a cotransforming T-DNA. C, Exchange T-DNA pSDM3732 contains an additional loxP site near the right-border repeat. Arrows marked with a 2 indicate cassette exchange via a Cre-mediated circularization step of the exchange T-DNA prior to insertion at the target site (A) and resolution of the inserted exchange cassette. Circle formation can occur prior to or after random integration of pSDM3732. One of the two hypothetical exchange reactions is drawn for circularized exchange T-DNA pSDM3732 via insertion at the loxP site, followed by resolution at the lox5171 site. D, Result of precise cassette exchange (with either exchange construct pSDM3327 [B] or pSDM3732 [C]). The restored nptII selectable marker allows selection for correct cassette exchange using kanamycin. Large black arrows, loxP sites; large dashed arrows, mutant lox sites, lox5171; the direction of arrowheads in the boxes indicates promoter regions; small white triangles, A. tumefaciens left- and right-border repeats. For the Southern strategy, probes are drawn as gray boxes above the constructs; N, NsiI; P, PstI; S, SalI; X, XhoI; the expected fragment length (kb) is indicated above the lines. Beneath the constructs, white arrows indicate primer binding sites, the expected size of the PCR fragments is given, with the RMCE-PCR product from primers p1 and r4 indicated with a dashed line. E, Nucleotide sequence of the DE35S-loxP-bar fusion of the target cassette and the loxP-nptII fusion of the exchange cassette. Upon cassette exchange, nptII expression is initiated at the transcription and translation signals of the DE35S promoter, located outside the cassette. Restriction endonuclease recognition sites used for cloning are indicated. loxP sites are framed and their orientation is indicated with an arrow. The translational start codon ATG at the NcoI site for bar and nptII is framed.