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Fig. 1. Generation and characterization of R246S knock-in ES cells. (A) Schematic of the targeting strategy. Black boxes represent p53 exons (exon numbers are indicated above). The positions of the probes used for Southern blotting are indicated below the diagram. Floxed neomycin and DTA expression cassettes are also indicated. Restriction sites are indicated as follows: E, H, Bs and B represents EcoRI, HindIII, BssHII and BamHI, respectively. Expected fragment sizes for Southern screening are indicted by the arrows and the position of the R246S mutation is indicated with the arrowhead. WT, wild type; Rec, after Cre-mediated recombination. The small green triangle represents the single loxP site remaining after Cre-mediated recombination. (B) Results of Southern blot hybridization, using exon 11 as the internal probe (left panel) and exon 1 as the external probe (right panel), after EcoRI digestion are shown for one representative targeted clone (p53+/R246S-Neo) and one clone with the targeted- and neomycin-cassette removed (p53+/R246S). Arrowheads point to the position of the expected bands for the mutant allele. (C) Expression of the mutant p53 allele in ES cells was analyzed by RT-PCR-RFLP. ES cells of various genotypes were treated with 0.5 µg/ml doxorubicin (Dox.) for 3 hours and p53 transcripts were PCR amplified prior to digestion with BsrBI. Restriction fragments generated from the respective alleles are indicated with arrowheads. (D) Conformation of R246S mutant p53 protein was determined by immunoprecipitation using conformation-specific antibodies Pab240, which recognizes the mutant, and Pab246, which recognizes the wild-type conformation, followed by immunoblotting. H1299 cells expressing the R175H human p53 mutant were used as a positive control for mutant conformation. (E) Protein level of p53 and actin was determined by western blotting. (F) Localization of p53 was analyzed by fluorescence confocal microscopy. ES cells were mock (–
) or 20-Gy (+)-irradiated and harvested 3 hours later. Representative images are shown in which the green fluorescence represents p53 protein and red fluorescence represents genomic DNA.
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