Reproducible in vivo applications of the p16-3MR model in senescence research

The p16-3MR mouse model has been widely used to visualize and conditionally eliminate p16-expressing senescent cells in vivo and has been applied across diverse biological contexts, including tissue repair, fibrosis, cancer, therapy response, and aging. Despite the development of multiple senescence reporter and ablation systems, p16-3MR stands out for its broad and sustained adoption across independent laboratories. Here, we review the extensive published literature supporting the reproducibility and utility of the p16-3MR system and present new validation data across acute and chronic senescence-inducing conditions. We demonstrate reproducible induction of p16-associated bioluminescence during wound healing, chemotherapy, and aging, as well as partial but consistent reduction following ganciclovir treatment. We further delineate the strengths and limitations of the individual components of the 3MR construct, including HSV-thymidine kinase-mediated clearance, Renilla luciferase- based bioluminescence, and monomeric red fluorescent protein, and discuss how factors such as cell abundance, tissue context, pigmentation, and substrate chemistry influence detection sensitivity. Together, these data confirm that the p16-3MR model is a functional and versatile tool for studying senescent cells in vivo when used with appropriate experimental design and interpretation, and they provide support for its continued application alongside emerging senescence models.