Abstract
SUMMARYThe site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2–6 of the endogenous p53 gene in mice to generate a p53FRT allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-KrasG12D/+; p53FRT/FRT mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53FRT mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.
Highlights
The transformation related protein p53 gene, Trp53, is the most frequently mutated gene in human cancer, altered in approximately 50% of human malignancies (Brosh and Rotter, 2009)
Generation of p53FRT mice To generate a frted p53 mouse, we constructed a targeting vector in which exons 2 through 6 of p53 genomic DNA are flanked by FRT sites (Fig. 1A)
The 5Ј FRT site was inserted between exons 1 and 2, and a loxP-flanked PGK-neo cassette followed by a 3Ј FRT site was inserted between exons 6 and 7
Summary
The transformation related protein p53 gene, Trp, is the most frequently mutated gene in human cancer, altered in approximately 50% of human malignancies (Brosh and Rotter, 2009). Conditional recombination of p53 using the Cre-loxP system has been utilized to delete or mutate p53 in a tissue-specific manner or to delete p53 at a specific time during development (Donehower and Lozano, 2009). This technology has led to the development of multiple mouse models of primary cancer (Marino et al, 2000; Lin et al, 2004; Jackson et al, 2005; Kirsch et al, 2007; Martinez-Cruz et al, 2008). Despite the availability of other highly efficient recombinase-based approaches, mouse models of cancer initiated by recombinases have generally used Cre to mutate genes in tumor parenchymal cells, ruling out the possibility that Cre can be used to genetically modify tumor stromal cells
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