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. 2002 Apr;22(8):2441–2449. doi: 10.1128/MCB.22.8.2441-2449.2002

FIG. 1.

FIG. 1.

Generation of p53S23A ES cells. (A) The endogenous configuration of the p53 gene in AY ES cells. AY ES cells have one wild-type p53 allele and one mutant p53 allele (AY allele) with p53 exons 2 through 4 deleted. The initiation codon, ATG, of the p53 gene is located in exon 2, and the AY allele does not produce a truncated p53 protein. Open boxes represent the p53 exons, and the filled bars represent probes for Southern blot analysis. The wild-type 14-kbp EcoRI and 7-kbp HindIII fragments are indicated by arrows, as are the mutant 6-kbp EcoRI and 5.8-kbp HindIII fragments on the AY allele. (B) The targeting construct. ∗, the Ser23-to-Ala mutation in exon 2. The PGK-neor gene flanked by LoxP sites was inserted into an engineered SalI site within intron 4. (C) Targeted p53 locus after homologous recombination between the wild-type p53 allele and the targeting vector. The positions of the PCR primer sets that were used to screen for LoxP/Cre-mediated deletion are shown by arrowheads. The sizes of the mutant EcoRI and HindIII fragments are indicated. (D) Mutant p53 allele after the PGK-neor gene was deleted. The size of the mutant HindIII fragment after the PGK-neor gene was deleted is indicated. (E) Southern blot analysis of genomic DNA derived from the wild type (lane 1), AY ES cells (lane 2), and targeted AY ES cells (lane 3), in which homologous recombination had occurred between the germ line allele and the targeting vector. Genomic DNA was digested with EcoRI and hybridized to probe A. Both the targeted allele and AY allele yielded the same 6-kbp mutant EcoRI fragment. The positions of both germ line and mutant alleles are indicated with arrowheads. (F) Southern blot analysis of genomic DNA derived from wild-type ES cells (lane 1), targeted AY ES cells with the PGK-neor gene inserted (lanes 2 and 3), AY ES cells (lane 4), and p53Ser23Ala ES cells (lane 5). Genomic DNA was digested with HindIII and hybridized to probed B. The 7-kbp wild-type band, 7.1-kbp PGK-neor gene deleted band, 5.8-kbp AY mutant band, and 3-kbp PGK-neor gene inserted band as well as the 7.8-kbp band derived from the p53 pseudogene are indicated.