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Methylated cytosines within CpG dinucleotides (mCpGs) along the DNA-binding domain of the TP53 tumor suppressor gene (exons ~5-8) are the single most significant mutational target in human cancers. The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences spanning exons 4-9 in both copies of the mouse p53 gene. To date, however, the methylation status of cytosines within CpGs in the substituted segment of the Hupki mouse genome has not been determined. This lack of information deserves special attention because DNA methylation in mammals, which occurs almost exclusively within CpG dinucleotides, is a dynamic process throughout developmental stages and may vary among different species. Here, we have investigated the status of CpG methylation in the substituted segment of the Hupki mouse genome, and compared it to the methylation profile of the corresponding segment in the human genome using the combined bisulfite-restriction analysis and sodium bisulfite genomic sequencing. We found that all cytosines within CpGs of the TP53 DNA-binding domain, on both the coding and noncoding strands, were heavily methylated in Hupki fibroblasts, as they were in human fibroblasts. This is in keeping with the fully methylated status of TP53 CpGs that is known to prevail in adult human tissues. The remarkably similar patterns of cytosine methylation within CpG dinucleotides in Hupki cells and human cells further validates the suitability of mutagenesis assays in Hupki cells for experimental induction of TP53 mutations that have been observed in human tumors.