Figure 1. Design and Validation of the p16^LUC Allele.

(A) Schematic of the p16^LUC knockin targeting strategy. “+ Flp” denotes the targeted allele after Flp-recombinase-mediated excision of the neomycin selection cassette.

(B) Induction of p16^INK4a mRNA is shown in MEFs of indicated genotypes passaged on a 3T3 schedule. Fold induction was calculated with respect to p16^INK4a transcript levels at day 3. Data shown correspond to three biological replicates performed in triplicate. Error bars represent SEM.

(C) p19^ARF and p16^INK4a western blots for littermate MEFs cultured on a 3T3 schedule and harvested at the indicated time points.

(D) Fold p16^INK4a induction is shown for the western blots represented in (C). Bands were quantified by using a LICOR Odyssey system, normalized to total protein, and analyzed as in (B).

(E) Luciferase activity in MEFs of the indicated genotypes, with results calculated and presented as in (B). Error bars are ±SEM.

(F) Correlation of luciferase activity and endogenous p16^INK4a expression in p16^+/LUC MEFs passaged on a 3T3 schedule. Data represent ≥3 biological replicates at multiple in vitro time points with best-fit line and 95% confidence intervals (dashed lines) shown. Linear regression was used to calculate the p value and correlation coefficient (r).

(G) Representative, 2 min luminescent images of a lactating p16^+/LUC mouse at the indicated time points relative to the weaning date (WD).

(H) Representative, 2 min photographic and luminescent images of a p16^+/LUC mouse with healing wounds.

See Figure S1 for additional related data.