Tumor spectrum analysis in p53-mutant mice Tyler Jacks, Lee Remington, Bart O. Williams, Earlene M. Schmitt, Schlomit Halachmi, Roderick T. Bronson, Robert A. Weinberg  Current Biology  Volume 4, Issue 1, Pages 1-7 (January 1994) DOI: 10.1016/S0960-9822(00)00002-6

Figure 1 p53 gene targeting. (a) Scheme for targeting one allele of the murine p53 gene in ES cells. Homologous recombination between the p53KO targeting vector and one allele of the endogenous p53 gene results in the replacement of p53 coding sequences between exons 2 and 7 with the neo gene expression cassette and the formation of the p53Δ mutant allele. ES cell clones were screened by Southern blotting of genomic DNA digested with EcoRI (R) plus StuI (S) and hybridized to probe derived from p53 exon 1 (A), which is not present in p53KO. The structure of the mutant allele in putative heterozygous ES-cell clones was confirmed using a 3′ cDNA probe (B), covering exons 7–10. Details of the construction of p53KO are described in Materials and methods. (b) Genotypic analysis of offspring from a p53 heterozygous cross. Tail DNA was isolated from offspring at weaning and examined by Southern blotting as described above using probe A. This probe hybridizes to an 8.0 kb R-S fragment from the wild-type allele (wt) and a 6.5 kb R-S fragment from the p53Δ allele (mut). Animals 2 and 6 are p53Δ/Δ, animal 7 is p53+/+, and animals 1, 3, 4, and 5 are p53+/Δ. Molecular weight standards are shown (lane 1Kb), along with their sizes (in kb) at right. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 1 p53 gene targeting. (a) Scheme for targeting one allele of the murine p53 gene in ES cells. Homologous recombination between the p53KO targeting vector and one allele of the endogenous p53 gene results in the replacement of p53 coding sequences between exons 2 and 7 with the neo gene expression cassette and the formation of the p53Δ mutant allele. ES cell clones were screened by Southern blotting of genomic DNA digested with EcoRI (R) plus StuI (S) and hybridized to probe derived from p53 exon 1 (A), which is not present in p53KO. The structure of the mutant allele in putative heterozygous ES-cell clones was confirmed using a 3′ cDNA probe (B), covering exons 7–10. Details of the construction of p53KO are described in Materials and methods. (b) Genotypic analysis of offspring from a p53 heterozygous cross. Tail DNA was isolated from offspring at weaning and examined by Southern blotting as described above using probe A. This probe hybridizes to an 8.0 kb R-S fragment from the wild-type allele (wt) and a 6.5 kb R-S fragment from the p53Δ allele (mut). Animals 2 and 6 are p53Δ/Δ, animal 7 is p53+/+, and animals 1, 3, 4, and 5 are p53+/Δ. Molecular weight standards are shown (lane 1Kb), along with their sizes (in kb) at right. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 2 Immunoprecipitation of p53 protein. Fibroblasts isolated from wild-type (+/+), heterozygous (+/–), and homozygous mutant (–/–) embryos were labeled with a [35S] methionine and [35S] cysteine, and whole cell lysates precipitated with negative control (C) or two p53-specific antisera: pAb248 (2) and PAb421 (4). Position of molecular size markers are shown (in kD) on the right and the expected position of p53 protein on the left. Note the absence of a specific signal from –/– cells and that the signal from the +/– cells is reduced compared with +/+ cells. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 3 Effects of p53Δ mutation on survival. Viability of 34 wild-type animals (white circles), 100 heterozygotes (blue circles), and 49 homozygous mutants (red circles) was monitored for a total of 500 days. Points represent animals that had died or had to be sacrificed owing to ill health. The single wild-type animal that died during this period had developed a tumor. Tumors were also observed in 17 of 24 heterozygotes and 41 of 49 homozygotes examined by necropsy (see text). Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 4 Tumor distribution in heterozygous and homozygous mutant animals. Pie charts show the relative frequency of tumor types observed in p53+/Δ and p53Δ/Δ mice. Frequencies determined from 44 total tumors in heterozygotes and 56 total tumors in homozygotes. Note that the sarcoma is the most common tumor type in heterozygotes, whereas lymphomas are most common in homozygotes. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 5 Histopathology of representative tumors. (a) Thymic lymphoma from a p53Δ/Δ animal with tumor cells surrounding a blood vessel (BV). Arrows delineate the border of the tumor and normal lung (NL), which may contain infiltrating tumor cells. (b) Hair matrix tumor from a p53+/Δ animal consisting of regions of transformed basal cells (B) and (above) exfoliating squamous cells, ghost cells, and keratin. (c) Rhabdomyosarcoma isolated from a heterozygous animal showing tumor giant cells (filled arrows) and a characteristic strap cell (unfilled arrow). (d) Osteosarcoma from a p53+/Δ animal. Tumor consists of both fibroblastic (F) and osteoblastic (O) regions; a characteristic bone spicule is indicated (arrow) Magnification: panels (a) and (c), 300X; panels (b) and (d), 75X. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)

Figure 6 Loss of heterozygosity. Southern blot analysis of DNA isolated from tumors of p53+/Δ mice. DNA was digested with EcoRI and StuI and hybridized with probe B shown in Figure 1. Tail DNA (T) from a heterozygous animal shows three bands, which are derived from the wild-type p53 allele (wt), the p53Δ allele (mut) and the p53 pseudogene (ψ). Of the six tumor samples shown, five show a greatly reduced signal from the wild-type allele, consistent with its loss during tumorigenesis. One tumor (H) has retained the germline pattern. H, hepatoma; L, lymphoma; S, anaplastic sarcoma; He, hemangiosarcoma; O, osteosarcoma; F, fibrosarcoma. Current Biology 1994 4, 1-7DOI: (10.1016/S0960-9822(00)00002-6)