B6 cg tg nes cre 1kln j

Human samples from the National Disease Research Interchange and the Center for LAM Research at Brigham and Women’s Hospital were collected with IRBs’ approvals.
The animal studies approved by IACUC according to the NIH guidelines used B6.Cg-Tg(Nes-cre)1Kln/J, C57BL/6-Tg(Nes-cre/Esr1)1Kuan/J, Tsc1tm1Djk/J, B6.129P2-Trp53/J (The Jackson Laboratory), Hupki P72, and Hupki R72 mice (from Dr. Maureen Murphy). Tamoxifen in corn oil (40 mg/ml) was administered i.p. (120mg/kg/day) for two days.

Hsd11b2^f/f mice were generated on a C57BL6 background (Artemis Pharmaceuticals, Cologne, Germany) by inserting LoxP sites into intron 1 and 5. These mice were bred with transgenic mice expressing Cre recombinase under the control of a rat nestin promoter/enhancer (B6.Cg-Tg(Nes-cre)1Kln/J; Jackson Laboratory, USA), as we described.^{23 (link)} This generated Nestin-Cre.Hsd11b2^fl/fl offspring (Hsd11b2 Brain Knock-out; Hsd11b2.BKO) and Hsd11b2^fl/fl littermate controls. All experiments were performed blinded to genotype and in accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act, following ethical review by the University.

All animal procedures were approved by the Ethics Committee on the Use of Animals of the Institute of Biomedical Sciences, University of São Paulo (protocol number 12, approved on 03/19/2013). To induce neuronal deletion of Stat5a and Stat5b genes, the Nestin-Cre strain (B6.Cg-Tg(Nes-cre)1Kln/J, Jackson Laboratories) was bred with mice carrying loxP-flanked Stat5a/b alleles as previously described (Cui et al., 2004 (link); Lee et al., 2008 (link)). Mice carrying neuronal deletion of STAT5 were homozygous for the loxP-flanked Stat5a/b alleles and hemizygous for the Nestin-Cre transgene, whereas the control group was composed of littermate animals carrying only the loxP-flanked Stat5a/b alleles. Mice were weaned at 3–4 weeks of age and the genomic DNA was extracted from tail tip for genotyping through PCR using a commercially available kit (Sigma). To confirm the efficacy of the deletion, mice were perfused, as described later, 90 min after receiving an intraperitoneal (i.p.) injection of ovine prolactin (5 μg/g, Sigma). We assessed the ability of prolactin to induce pSTAT5-immunoreactivity (pSTAT5-ir) in the brain of control and N-STAT5 KO females. In addition, the hypothalamus of an additional group of control and N-STAT5 KO females was collected to determine the expression of STAT5a and STAT5b mRNA levels.

Mice were housed in a temperature, humidity and light-controlled facility on a 12 h light/dark cycle and given free access to food and water. Experiments were performed in age-matched controls of both sexes, divided as evenly as possible between males and females. Lrrc8a^fl/fl (Swell1^fl/fl) mice were generated as previously described.^{23 (link)} Brain-specific Lrrc8a knockout mice were produced by breeding Lrrc8a^fl/fl female mice with commercially available Nestin^Cre/+ male mice (B6.Cg-Tg(Nes-cre)1^Kln/J; Jackson Laboratory stock #003771, RRID: IMSR_JAX:003771), both on a C57BL/6J background. Nestin^Cre/+;Lrrc8a^fl/+ heterozygous males were crossed again with Lrrc8a^fl/fl female mice to produce Nestin^Cre/+;Lrrc8a^fl/fl knockout mice and littermate controls. Four possible genotypes, Lrrc8a^fl/+ (abbreviated fl/+), Lrrc8a^fl/fl (fl/fl), Nestin^Cre/+;Lrrc8a^fl/+ (heterozygous deletion, Het), and Nestin^Cre/+;Lrrc8a^fl/fl (constitutive knockout, KO) were confirmed by PCR analysis. Lrrc8a deletion was verified by PCR amplification across the predicted loxP insertion sites surrounding Lrrc8a exon 3. The Nestin^Cre transgene insertion into mouse chromosome 12 was validated according to the Jackson Laboratory genotyping protocol.