Figure 2

E2f3 expression may be reversibly induced in E2f3 ^TRE/TRE mice. (a) Schematic of rtTA driven expression from the E2f3 ^TRE locus. Grey box- exon, blue box- TRE element, green oval- reverse tetracycline trans activator, orange triangle- doxycycline. (b) Quantitative RT-PCR analysis of E2f3 expression in adult control (n = 6(spleen, lung), n = 5(liver)), E2f3 ^TRE/TRE; Rosa26 ^rtTA/+ (n = 4) and E2f3 ^TRE/TRE; Rosa26 ^rtTA/rtTA (n = 3) mouse tissues following administration of doxycycline for three days. Expression is normalized to HPRT and relative to the mean of the control samples. Error bars, s.e.m. Two-way ANOVA with a Tukey’s multiple comparisons test; *P < 0.5, ***P < 0.001. (c) Immunoblot analysis of E2F3 protein levels in adult control, E2f3 ^TRE/TRE; Rosa26 ^rtTA/+ and E2f3 ^TRE/TRE Rosa26 ^rtTA/rtTA mouse tissues following administration of doxycycline for three days. Replicate samples are generated from tissues isolated from independent mice. Expression of GAPDH is included as a loading control. Refer to Supplementary Fig. 11 for full images. (d) Quantitative RT-PCR analysis of E2f3 expression in adult control (n = 3) and E2f3 ^TRE/TRE; Rosa26 ^rtTA/+ tissues isolated from mice following administration of doxycycline for three days only (+3, n = 2) or for three days with subsequent withdrawal for seven days (+3/−7, n = 5). Expression is normalized to HPRT and relative to the mean of the control samples. Error bars, s.e.m. Two-tailed t-test: control vs. +3/−7, P = 0.82 (spleen), 0.10 (liver), 0.52 (lung); n.s., not significant. Control genotypes include E2f3 ^TRE/TRE in the presence and absence of doxycycline and E2f3 ^TRE/TRE; Rosa26 ^rtTA/+ and E2f3 ^TRE/TRE Rosa26 ^rtTA/rtTA maintained in the absence of doxycycline.