Figure 2  Generating ES cells with a p53 RMCE-ready locus. The p53 gene is contained in a 17 kb-long EcoRI (RI) fragment (black boxes: coding sequences, white boxes: UTRs). The Flox targeting construct (below), the sequence which was verified before use (Materials and Methods), contains (i) a 3.4 kb-long 5′ homology region; (ii) 0.2 kb upstream of coding sequences, an EcoRI site and L3, a mutant loxP [loxP257, (14)]; (iii) p53 exons; (iv) 0.4 kb downstream, a puroΔTK fusion gene (puDTK) for positive/negative selection (21) and an inverted WT loxP (1L); (v) a 1.2 kb-long 3′ homology region and (vi) the diphteria α-toxin (DTA) gene for targeting enrichment. The recombinants resulting from the depicted crossing-overs are identified by a 6.5 kb band in Southern blot with probe A and a 3 kb band by PCR with primers a and b. A representative Southern, and PCR of one positive (β) and two negative clones, are shown.