Nozomi Hori, Shimpei Kawamoto, Ken Uemura, Yumi Kinugasa‐Katayama, Yumiko Okumura, Kentaro Tanaka, Jeong Hoon Park, Masahiro Wakita, Daisuke Motooka, Naoko Ohtani, Eiji Hara
promoter, has been widely used to visualize and ablate senescent cells in vivo, but our analyses revealed critical limitations. Bioluminescence signals in p16-3MR mice were extremely weak and virtually indistinguishable from those of wild-type mice injected with coelenterazine-h, indicating that previously reported signals largely reflected substrate background rather than authentic reporter expression. Signal intensity remained unchanged with aging, doxorubicin treatment, or cutaneous wound healing, failing to replicate earlier observations. Furthermore, RFP signals were undetectable in senescent fibroblasts from p16-3MR mice, and senescent cells were not eliminated by ganciclovir treatment, suggesting poor expression and lack of functional activity of the mRFP and HSV-TK transgenes. These results demonstrate functional deficiencies in all three transgenes, highlighting the importance of using wild-type controls and calling for careful reevaluation of studies employing this system.