A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo

Abstract

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are suboptimal in vivo because of poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled noninvasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol- or CCl 4 -induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

Targeted cancer therapy induces APOBEC fuelling the evolution of drug resistance

“…Tumour burden in each animal was quantified by calculating the tumour volume of visible tumours using AnalyzeDirect. Autostainer Link 48 (Agilent, UK) as previously described 89 . Briefly, formalin-fixed paraffin embedded (FFPE) sections were dewaxed and subjected to heat induced epitope retrieval (HIER) on-board using a Dako PT module.…”

In vivo RNA-seq and ChIP-seq analyses show an obligatory role for the C terminus of p53 in conferring tissue-specific radiation sensitivity

“…Consistent with these findings, an engineered mouse strain with a fluorescent reporter that detects endogenous p53 activity revealed robust liver p53 activity in response to radiation. 72 …”

p53 and TIGAR promote redox control to protect against metabolic dysfunction-associated steatohepatitis

“…p53 FL/FL ( Trp53 tm1Brn ), Albumin -Cre (Speer6-ps1 Tg(Alb-cre)21Mgn ), PG13-iRFP ( Hprt Tm1(p53RE-iRFP [pg13])Bea ), and Tigar -/- mice were described previously 49, 50, 66, 67 and were fully backcrossed to C57BL6/J (N10). Lep ob (B6.Cg-Lep ob /J) and Lepr db (BKS.Cg- Dock7 m +/+ Lepr db J) heterozygous mice were purchased from Charles River (Strain codes: 606 and 607).…”

“…These observations suggest a potential role for p53 in mediating the aetiology of diet-induced liver disease. We have previously utilised a p53 reporter mouse to non-invasively monitor the p53 pathway after total body gamma-irradiation and during hepatotoxin-mediated liver regeneration 50 . In p53 reporter mice, active p53 induces expression of the near-infrared fluorescent protein iRFP713 (iRFP) that is linked to the synthetic PG13 p53-responsive promoter (Fig.…”