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. 2000 Apr 11;97(8):4174–4179. doi: 10.1073/pnas.97.8.4174

Figure 1.

Figure 1

The generation of a mouse containing a p53R172H substitution. (a) The genomic organization of the p53 gene and the targeting vectors are shown. The first recombination step results in incorporation of the neo and tk genes (PGKneoNTRtkpA) at the p53 locus. In the second step, the neo and tk genes are replaced with the genomic p53 containing a point mutation (asterisk), resulting in an arg-to-his substitution at amino acid 172. Δg represents the deletion of a G nucleotide at the intron 2 splice acceptor site. (b) Southern hybridization of EcoRI-digested DNA from several ES cell clones electroporated with the PGKneoNTRtkpA targeting vector. The 17-kb EcoRI fragment represents the wild-type p53 allele whereas the 3.4- and 12-kb EcoRI fragments are the expected sizes for the mutant. The ES cell clone (3C12) in lane 2 is correctly targeted. (c) SSCP analysis using primers spanning the mutation. Lanes: 1, plasmid containing wild-type p53 genomic sequences; 2, plasmid containing mutant p53 genomic sequences; 3, genomic DNA from targeted ES cell clone; 4, genomic DNA from normal ES cells. *, wild-type bands; **, mutant-specific bands. (d) Southern blot hybridization of EcoRI-digested DNA from derivatives of 3C12 cells targeted with a construct containing a missense mutation in p53. Lanes: 1, normal ES cell DNA; 2, 3C12 DNA; 3, DNA from a correctly targeted ES cell clone.