A19
Development of a novel in vivo dual fluorescence p53 activity reporter system
Kerryanne Crawford, Mark Boyd
University of Liverpool, Merseyside, UK
TP53 is the most commonly mutated gene in all human cancers [Vogelstein B., Nature, 1990] and co-ordinates many of the cellular responses to DNA damage. It is perhaps surprising therefore that there have been relatively few p53 reporter mouse strains described. To date, these have used non-targeted, largely artificial, repeated p53 response elements in their reporter constructs which are known to be associated with fidelity problems including those from positional effects. Thus it is unlikely that any of these truly provide authentic report of p53 activity. The ideal solution would be to target a p53 responsive gene using a knock-in approach, preferably in a gene that was exclusively activated by p53 and which also does not display any haploinsufficient phenotype. Whilst the latter may not be completely achievable we have set out to generate two reporter strains that would enable differential monitoring of p53 activation leading to cell cycle arrest and/or apoptotic responses.
In order to achieve our goal we must select reliable downstream p53 responsive genes in our tissue of interest, the gastrointestinal tract. In the case of cell cycle arrest p21 (Cdkn1a) was an obvious candidate gene, since it is essential for G1 phase p53 arrest and is readily up-regulated by DNA damage in the gastrointestinal tract ([Wilson J.W. et al., Am. J. Pathol, 1998] and our own data). However, in the case of apoptosis, there are many p53 target genes which could be up-regulated which made it imperative to review in detail the potential roles of many target genes including Bax, Noxa, Bid and Puma in the GI tract. Our analysis revealed that whilst Noxa may not be an ideal reporter, Puma appears to play a major role in inducing apoptosis in these tissues [Qiu W., et al., Cell Stem Cell 2008]. Accordingly, we are generating constructs to knock-in fluorescent proteins into these loci. In the first instance we are using mCherry and eGFP as proof of principal, but if these are successful then we will develop longer wavelength reporters to permit improved deep tissue imaging.
As visual technology advances these mice could also be used in real-time p53 responses to DNA damage or other stimuli, as well as for long term monitoring of gene-regulation during cancer development.
