Generation of mice for evaluating endogenous p16^Ink4a protein expression

The cyclin-dependent kinase inhibitor p16^Ink4a 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^Ink4a in tissues remain poorly understood, which hinders uncovering the role of p16^Ink4a 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^INK4A barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16^Ink4a is HA-tagged at its N-terminus, to explore the protein expression of p16^Ink4a at the organismal level. p16^Ink4a 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^Ink4a 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^Ink4a proteins induced. These results in our mouse model highlight the need for caution in evaluating p16^Ink4a protein expression in vivo.