Generation of p53 knock-in mice and cells

         A complete understanding of p53 relies on studying its function in vivo, where its activity in tumor suppression can be analyzed. To test the role of specific biochemically-defined activities of p53 in tumor suppression, we construct "knock-in" mouse strains in which the wild-type p53 gene is replaced with particular p53 mutant alleles. Besides examining the ability of these alleles to act as tumor suppressor genes in vivo, cells from these mice can be studied for their properties in classical apoptosis and transformation assays. Testing p53 function in this way, by expressing it at physiological levels, should provide definitive information about its mechanism of action, both as a tumor suppressor and in other p53 functions.

         One area of particular interests in understanding the role of p53-dependent transcriptional activation in its biological function in vivo. To address this question, we have generated mouse strains carrying mutations in the p53 transactivation domain. One of these mouse strains was engineered with two mutant residues in the transactivation domain, at codons 25 and 26, alterations that had been shown previously to render p53 hypomorphic as a transactivator. The initial characterization of the mouse strain expressing this mutant, termed p5325,26 (also known as p53QS), has provided us with a greater understanding of p53 function at the cellular level. While p5325,26 is compromised in its ability to activate the transcription of many p53 target genes, it does retain wild-type transactivation capacity on a subset of p53 targets, including the pro-apoptotic gene Bax, suggesting that p53 regulates gene expression via distinct, target–specific mechanisms. In response to DNA damaging agents, this mutant is only partially active in eliciting a G1 cell cycle arrest, corroborating previous studies by our lab and others which had shown, in an in vitro context, that transactivation is critical for the arrest function of p53. Strikingly, this p53 mutant shows stress-specific apoptotic activity. In oncogene-expressing MEFs treated with DNA damage, p5325,26 is completely defective at inducing apoptosis. However, in response to serum deprivation, this mutant retains partial apoptotic activity, and intriguingly, upon exposure to a hypoxic environment, p5325,26 induces significant levels of apoptosis. The finding that this mutant protein has stress-specific apoptotic capacity suggests that p53 acts through distinct pathways to induce apoptosis in response to different stimuli. Currently, we are evaluating the activity of this mutant in several in vivo settings previously shown to be sensitive to p53-dependent apoptosis to determine whether it displays cell type-specific apoptotic activity. We are also in the process of evaluating the ability of p5325,26 to suppress tumorigenesis in several models of cancer, studies that will significantly enhance our understanding of the role of transactivation by p53 in the prevention of tumor growth. In addition, we have created several additional knock-in mouse strains that are currently under investigation.

Targeting scheme used to generate p53 knock-in mice. The mutation of interest, along with a transcriptional stop cassette to allow expression of the mutant allele upon introduction of the Cre recombinase, are inserted into the mouse genome.
Northern blot analysis demonstrates that the p53-25,26 mutant is highly compromised in transcriptional activation of most p53 target genes, with the exception of Bax.


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