Figure 2.

Gene Replacement and Survival of Tsc1-Floxed Mice Injected with AAV-Cre Vectors with and without AAV-Hamartin Vectors, Including Body Weight and Rotarod Assessments

(A) Homozygous Tsc1-floxed pups received i.c.v. injection into both cerebral ventricles at P0 using the AAV1-Cre vector. At P21 in four separate experiments, mice were randomly assigned into three groups for retro-orbital injections (group 1 = mice injected with AAVrh8-hamartin [n = 12]; group 2 = mice injected with AAVrh8-GFP [n = 12]; group 3 = no injection [n = 5]). Log-rank (Mantel-Cox) survival curves were 156, 46, and 47 days for groups 1, 2, and 3, respectively. A highly significant difference, p < 0.0001 (log-rank test; GraphPad Prism), was found when comparing group 1 with groups 2 and 3. (B) Homozygous Tsc1-floxed pups received i.c.v. injection into both cerebral ventricles at P0 using the AAV1-Cre vector. At P21 days, in three separate experiments mice were randomly assigned to three groups for retro-orbital injections (group 1 = AAV-Cre + AAV9-hamartin [n = 13]; group 2 = no injection [n = 13]). Another set of mice (group 3) was injected with only AAV9-hamartin at P21 days with no Cre injection (n = 6). Mean survival, based on log-rank (Mantel-Cox) survival curves, in the different groups was 429, 32, and >450 days for groups 1, 2, and 3, respectively. Black arrow indicates when mice were sacrificed. A highly significant difference, p < 0.0001 (log-rank test, GraphPad Prism), was found when comparing groups 1 and 2. Groups 1 and 3 were also significantly different, p < 0.005. (C) Tsc1-floxed mice injected with AAV1-Cre vector with (n = 7) and without (n = 6) AAV9-hamartin vector showed normal weight gain as compared with naive mice (n = 5). (D) At an accelerated speed of 4–64 rpm for the rotarod test, the motor function of the Tsc1-floxed mice rescued by AAV9-hamartin vector was similar to the control and significantly decreased as compared with the AAV1-Cre-only group. ***p < 0.001; ****p < 0.001 (two-way ANOVA test).