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Temporal dissection of p53 function in vitro and in vivo

Abstract

To investigate the functions of the p53 tumor suppressor, we created a new knock-in gene replacement mouse model in which the endogenous Trp53 gene is substituted by one encoding p53ERTAM, a p53 fusion protein whose function is completely dependent on ectopic provision of 4-hydroxytamoxifen. We show here that both tissues in vivo and cells in vitro derived from such mice can be rapidly toggled between wild-type and p53 knockout states. Using this rapid perturbation model, we define the kinetics, dependence, persistence and reversibility of p53-mediated responses to DNA damage in tissues in vivo and to activation of the Ras oncoprotein and stress in vitro. This is the first example to our knowledge of a new class of genetic model that allows the specific, rapid and reversible perturbation of the function of a single endogenous gene in vivo.

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Figure 1: Generation of the Trp53KI allele and genotype analysis of Trp53KI/KI mice.
Figure 2: 4-Hydroxytamoxifen (4-OHT) restores the p53-mediated damage response in Trp53KI/KI mouse tissues in vivo and cells in vitro.
Figure 3: 4-Hydroxytamoxifen (4-OHT) restores the p53-mediated transcriptional regulation in Trp53KI/KI mouse tissues in vivo and cells in vitro.
Figure 4: The Trp53KI/KI system is competent for deactivation of p53ERTAM.
Figure 5: Persistence of DNA damage–induced p53 activating signal.
Figure 6: Response to Ras deregulation is dependent on 4-hydroxytamoxifen (4-OHT) and reversible.
Figure 7: 4-Hydroxytamoxifen (4-OHT) determines the immortalization, senescence and ploidy states of primary p53ERTAM cells.
Figure 8: Late-passage, but not early-passage, cultured primary fibroblasts activate p53 in the absence of any overt DNA damage or stress.

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Acknowledgements

We thank L. Heath for help with interpretation of the pathology of the Trp53KI/KI tumors; P. Rodriquez-Viciana for the H-ras V-12 retroviral vector; T. Littlewood for the p53minER vector; I. Rosewell for electroporation of ES cells; F. Rostker for assistance with animals; D. Ginzinger for Taqman analyses; A. Finch for assistance with histology and imaging; and F. McCormick, K. Shannon, D. Green, T. Tlsty, K. Vousden, R. Treisman, C. O'Shea, D. Iacovides and the members of the laboratory of G.I.E. for discussions, advice and criticism. This work was supported by grants from the US National Institutes of Health and by the University of California San Francisco Comprehensive Cancer Center Cell Cycle and Signaling Disregulation Program. D.M.-Z. was supported by grants from the Spanish Ministry of Science and Education. We dedicate our work to the memory of Stanley Korsmeyer.

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Christophorou, M., Martin-Zanca, D., Soucek, L. et al. Temporal dissection of p53 function in vitro and in vivo. Nat Genet 37, 718–726 (2005). https://doi.org/10.1038/ng1572

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