Fig. 1.

Conditional inactivation of the Tsc1 gene in embryonic brain results in postnatal lethality, decreased weight, increased mTORC1 and decreased mTORC2 signaling. (A) Schematic of Tsc1 gene targeted by Cre recombinase. (B) Cre mediated recombination of the Tsc1 gene in dorsal cortex microdissected from E13.5 embryonic brain. Genotypes: 1. Tsc1 Flox/Flox; Cre negative, 2. Tsc1 Flox/wt heterozygote; Cre positive, 3. Tsc1 Flox/Flox homozygote; Cre positive (Tsc1^Emx1-Cre CKO). Upper (closed) arrowhead indicates PCR product (2012 bp) of the unrecombined Tsc1 floxed allele, lower (open) arrowhead indicates PCR product (361 bp) from the recombined, inactivated Tsc1 allele. (C) Western blot of dorsal cortical extracts from representative P5 control and Tsc1^Emx1-Cre CKO mice, n=4 for each group. Decreased hamartin and tuberin expression in seen as well as increased mTORC1 and decreased mTORC2 signaling with increased levels of phospho-S6 (Serine235/236) and decreased phospho-Akt (Serine 473) respectively. Changes in baseline levels of S6 or Akt were not seen. GFAP is also increased in the dorsal cortex from Tsc1^Emx1-Cre CKO mice as compared to control littermates (C, also see Fig. 5B). Specific GFAP levels are seen by the lower two bands at 55 and 48 kD, with an upper non-specific band. Actin expression was used as a loading control. (D) Complete mortality of Tsc1^Emx1-Cre CKO mice by postnatal day 25 compared to littermate controls. Log-Rank test, p<0.0001. (E) Decreased postnatal weight in Tsc1^Emx1-Cre CKO mice (n=4) compared to littermate controls (n=9), error bars indicate standard deviation, asterisk denotes statistical significance using Students t-test p<0.05.