Generation of mice for evaluating endogenous p16 protein expression.

The cyclin-dependent kinase inhibitor p16 plays a central role in cellular senescence in vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16 in tissues remain poorly understood, which hinders uncovering the role of p16 under the in vivo context. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16 barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16 is HA-tagged at its N-terminus, to explore the protein expression of p16 at the organismal level. p16 was induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16 was not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16 proteins induced. These results in our mouse model highlight the need for caution in evaluating p16 protein expression in vivo.