Targeting construct for a p53 RMCE-ready locus
Details for plasmid construction are available upon request owing to space limitations mandating a brief outline. We started from a plasmid L3-1L containing heterologous loxP sites (L3 is the mutant loxP257 recently described (14), 1L is an inverted WT loxP). The WT loxP and loxP257 differ in their spacer sequences: the spacer sequence is 5′-ATGTATGC-3′ for WT loxP and 5′-AAGTCTCC-3′ for loxP257. The three mutations in the loxP257 spacer sequence prevent it to recombine with WT loxP, ensuring efficient RMCE in several cell lines: accurate RMCE with these loxP sites occurred with an average frequency of 81% at two loci in CHO cells and an average frequency of 69% at four loci in Hela cells (14). The L3-1L plasmid was first modified to include a ClaI and a FseI site between the LoxP sites, leading to plasmid L3-CF-1L. We next modified a puroΔTK plasmid (YTC37, a kind gift from A. Bradley) by using oligonucleotides to destroy a NotI site downstream of the puroΔTK gene and introduce a NotI site upstream, and a FseI site downstream of the gene (leading to plasmid CN-PuroΔTK-F). Next, a PmlI-MfeI 6.3 kb fragment from Trp53 was subcloned in a modified pBluescript KSII+ (pBS, Stratagene), and the resulting plasmid was digested with SwaI to introduce an EagI site, leading to p53PmlEag, a plasmid containing exons 2–11 of p53. We then inserted a 5.5 kb ClaI-EagI fragment from p53PmlEag in plasmid CN-PuroΔTK-F digested by ClaI and NotI, and inserted the resulting fragment between the loxP sites of L3-CF-1L by ClaI and FseI digestion, leading to L3-p53PmlEagPuroΔTK-1L. We next engineered a plasmid containing the region for 3′-homology downstream of the p53 gene and the DTA gene in two steps: (i) we performed a three-way ligation between a modified pBS digested by HindIII and NotI, a HindIII-EcoRI fragment from Trp53 for 3′homology and an EcoRI-NotI fragment containing the DTA gene, from plasmid pgkdtabpa (kind gift of P. Soriano), leading to plasmid 3′ + DTA and (ii) because the Bsu36I-EcoRI region downstream of p53 contains repetitive sequences (F. Toledo and G. M. Wahl, unpublished data), we later deleted this region, to obtain plasmid 3′ + DTA. The 5′ homology consists of a 3.4 kb-long BamHI-PmlI fragment from intron 1 of p53 cloned in a modified pBS (plasmid p5′). Finally, appropriate fragments from plasmids p5′, L3-p53PmlEagPuroΔTK-1L, and 3′ + DTA were assembled in a modified pSP72 plasmid (Promega). Plasmid Flox, the resulting targeting construct, was verified by restriction analysis, then sequenced using 30 primers chosen to precisely verify all p53 coding sequences, all exon–intron junctions and the sequences at and around the loxP sites.