The long-term objectives of this proposal are to test a humanized gene-targeted p53 tumor suppressor gene knock-in mouse as an experimental tool for molecular epidemiology, chemoprevention, and cancer therapy studies. This new model is designed to reflect and predict more faithfully than the currently used mouse models, human in vivo mutagenesis, carcinogenesis and responses to therapeutic drugs that target p53 protein.
The specific aims are 1) to induce tumor mutations in human p53 sequences of knock-in mice exposed to human carcinogens, and compare them to human tumor p53 mutation spectra; 2) to test whether a p53 hotspot mutation common in aflatoxin-associated human liver cancers occurs in liver tumors of aflatoxin B1-exposed, hepatitis B virus-transgenic/human p53 knock-in mice; 3) to generate mouse strains that harbor a mutant human p53 sequence identical to one of the inherited p53 mutations of cancer-prone Li-Fraumeni families. These p53 mutant mouse strains provide a new animal model, with an allelic configuration that parallels the human situation, for testing chemotherapeutic agents and chemopreventive dietary factors; 4) to replace mouse regulatory sequences with human elements, and determine how this alters p53 expression; these data may be useful in developing next generation p53 knock-in mouse strains. The research plan involves: 1) Construction of gene-targeting plasmids; 2) Generation of gene-targeted mouse strains in which exons 5-9 of the endogenous mouse p53 gene have been replaced by the corresponding human segment; 3) Treatment of p53 knock-in strains with human carcinogens; 4) Analysis of tumors and normal tissues for p53 mutations, and alterations in p53 message and protein. The methods to be used are: (a) Standard cloning procedures for plasmid construction; (b) gene-targeted technology with ES cells, and knock-in/knock-out strategies; (c) mouse breeding and treatment with carcinogens; (d) DNA sequencing, PCR and RT-PCR amplification; (e) immunohistochemistry; (f) mutation analysis by DGGE and allele-specific PCR. New pharmaceuticals designed to regenerate tumor suppressor functions of a mutated p53 in humans can be tested in vivo in these humanized"""""""" mice because they have a core domain identical to the human p53 protein rather than the mouse p53 protein.
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