Figure 1.

Generation of p53 point mutated alleles in ES cells. Scheme for targeting one allele of the murine p53 gene in ES cells with a targeting vector containing either the R270H or the P275S mutation (asterisk), and a neo-TK selectable marker cassette flanked by LoxP sites (first selection round). Homologous integration of the vector results in an additional EcoRI site (situated in the promoter of pMC-neo), which is used for Southern blot analysis of neomycin-resistant ES cell clones. In the homologous recombinant clones excision of the neo-TK selectable marker cassette was accomplished by transfection with circular pMC-CreN plasmid (second selection round). The resulting allele differs from the wild-type allele, besides the R270H or P275S mutation, only in the presence of one LoxP site downstream of the coding sequences.