Chronic Transgene Suppression and Arrest of Aβ Aggregate Formation in an Independent Line of Tet-Off APP Mice (CaMKIIα-tTA × tet-APPswe/ind Line 18)
(A) The experiment presented in the text for line 107 tet-off APP was repeated with a second tet-off APP line (line 18) to control for integration site artifacts. Cortical homogenates from untreated control and dox-treated double transgenic mice were immunoblotted for full-length APP with the human-specific antibody 6E10 to confirm transgene suppression at the time of harvest. Immunostaining for endogenous superoxide dismutase (SOD1) was included as a loading control.
(B) Quantitation of signal intensity from the Western blot in (A) shows transgenic APP levels in line 18 are suppressed by more than 98% following 3 mo of dox treatment (significant effect of group ANOVA F 2,8 = 1559.7, p < 0.001). This level of suppression was equal to or better than that attained in line 107 (see Figure 3B).
(C) Serial dilution filter trap assay was used to quantify aggregated Aβ in cortical homogenates.
(D) Quantitation of signal intensity in the linear range of the dilution series shown in (C). Consistent with the amyloid histology shown in Figure S5, aggregate formation was significantly increased between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA F 2,18 = 12.14, p < 0.001). Aggregate formation was completely arrested by transgene suppression, and is identical in 9-mo-old mice treated with dox for 3 mo as in untreated animals harvested when treatment began (p > 0.5, Tukey post-hoc test). *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test.
(962 KB TIF).