Animals and tamoxifen administration

Care and use of all mice were conducted according to an animal protocol approved by the Washington University Animal Studies Committee (IACUC #A-3381-01) and consistent with NIH guidelines on the Care and Use of Laboratory Animals, the ARRIVE guidelines, and the Basel Declaration. In addition, NIH guidelines on Rigor and Reproducibility in Preclinical Research were followed, including use of randomization, blinding, both sexes, and statistical/power analyses. GFAP-CreER mice (obtained from Suzanne Baker’s lab)²³ were crossed with Tsc1^flox/flox mice to generate the inducible Tsc1 knockout mice (Tsc1^GFAP-CreER mice). Littermates (Tsc1 f/+ ;CreER+ or Tsc1 f/f; CreER-) were used as negative controls. For comparison, conventional Tsc1^GFAP-Cre mice were also generated by crossing non-inducible GFAP-Cre mice with Tsc1^flox/flox mice ¹⁵. In separate studies, CgGt(Rosa)26Sor^{tm6(CAG-ZsGreen1)Hze}/J (Rosa-Green; Jackson Laboratory) reporter mice were crossed with GFAP-CreER and GFAP-Cre mice to confirm the cellular localization of Cre-mediated recombination induced by tamoxifen.

Mice were randomized into tamoxifen or vehicle treatment groups. Tamoxifen (Sigma, T5648) was dissolved in corn oil (Sigma, C8267) at a concentration of 20 mg/ml. Tamoxifen (9mg/40g body weight; i.p.) was administered to mice once a day for 5 consecutive days at 6-weeks or every other day at 2 weeks of age.