Supplementary Figure 2: Step-wise Cre recombination and inversion of the FLIP cassette.
From: One-step generation of conditional and reversible gene knockouts
(a) Inversion (flipping) of the FLIP cassette. Schematic showing the step wise recombination of loxP sites following Cre treatment. In the first recombination the loxP sites represented by pink triangles (left) or the loxP sites represented by purple triangles (right) will be recombined. Due to the orientation of the loxP sites this results in an inversion. During the second recombination the loxP sites, now aligned in the same direction recombine. The result is deletion of the PGK promoter and branch point 1 (BP1). SD, splice donor; SA, splice acceptor, BP, branching point; purple triangles, LoxP1 sites; Pink triangles, Lox5171 sites.
(b) Genotyping strategy used to confirm clones targeted with the FLIP cassette. The arrows represent primers, and the primer pairs are colour coded. The drawing indicates the position of the primers in the genome and in the FLIP cassette. The blue and orange primer pairs were used to confirm correct integration of the FLIP cassette. The allele not having an integrated a FLIP cassette but potential sustained indels due to NHEJ is genotyped and sequenced with the primer pair represented by the green arrows.
