129s1 svimj

The CC founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility at the Helmholtz Centre, Braunschweig for two to six generations depending on the strain. All mice were maintained under specific pathogen free conditions and according to the German animal welfare law. At the HZI, mice were housed in IVC cages (Techniplast Sealsafe, Typ 1284L) and paper tissues as cage enrichments with a light–dark cycle of 14 h/10 h without changes to summer savings time. Mice were fed standard diet (Ssniff V1534-300). At the GMC mice were housed in individually ventilated caging (IVC) systems (IVC System Green Line, Tecniplast, Italy), with a 12/12 h light–dark cycle, and red houses as cage enrichment. The IVCs operate at positive pressure. Mice were fed with irradiated standard and breeding rodent diet (Altromin 1314) ad libitum unless indicated otherwise. At 7 weeks of age, up to five cohorts with about four animals per sex and strain were shipped to the Helmholtz Zentrum Munich. Mice were acclimatized for 2 weeks before testing started at 9 weeks of age.

Thirteen-day-old embryos of Sirt-3 WT (129S1/SvImJ, Stock No: 002448 Jackson Laboratory, Bar Harbor, ME, USA) and Sirt-3 KO (Stock No: 012755, Jackson Laboratory, Bar Harbor, ME, USA) female mice were used for isolation of male and female mouse embryonic fibroblasts (MEFs) according to [86 (link)], with the modification that individual embryos were isolated to enable sex-based differentiation. Cells were maintained in a 37 °C incubator with 5% CO₂ and immortalized by stable transfection with SV40 T-antigen-containing plasmid. Sex determination of MEFs was performed by real-time PCR analysis utilizing specific primers targeting the sex-determining region Y (Sry) gene on the Y chromosome. For both RNA extraction and Western blots, low-passage cells were used.

Seven months old male PPARα KO mice (129S4/SvJae-Pparα^tm1Gonz/J) and corresponding WT (control) mice (129S1/SvImJ) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were kept in Macrolon cages in a room maintained with controlled temperature (23±1°C), humidity (50–60%), and lighting (6.00 a.m to 6.00 p.m). 32 mice of each genotype with an average initial body weight of 29.0±2.3 g (WT mice) and 28.8±2.1 g (PPARα KO mice) (mean ± SD, n = 32) were randomly assigned to two groups of 16 mice each. One group (“fed”) of each genotype received the commercial diet for rodents (“altromin 1324”, Altromin GmbH, Lage, Germany) ad libitum for the next 48 h (fed WT mice, n = 16; fed PPARα KO mice, n = 16), whereas from the other group (“fasted”) the diet was removed and mice were fasted for the next 48 h (fasted WT mice, n = 16; fasted PPARα KO mice, n = 16). Water was available ad libitum from nipple drinkers during the experiment. The study was approved by the respective regional government agency of Saxony-Anhalt (“Landesverwaltungsamt”, approval number H1-4/T1-10). All efforts were made to minimize suffering. Mice were then killed by decapitation under light anaesthesia with diethyl ether.

The Thomas Jefferson University (TJU) Institutional Animal Care and Use Committee have approved these studies. All methods were performed in accordance with the guidelines and regulations of TJU Institutional Biosafety Committee. Reporting of the animal experiments followed the recommendations in the ARRIVE guidelines. C57BL/6J, 129S1/SvImJ, BALB/cJ, NOD/ShiLtJ and CAST/EiJ were purchased from The Jackson Laboratory (Bar Harbor, ME). All CC strains used were purchased in 2014–2015 from the Systems Genetics Core Facility, University of North Carolina at Chapel Hill (Chapel Hill, NC). Mice were housed in micro-isolator cages with free access to food and water and were maintained and bred in a specific pathogen-free facility at TJU. Three-week-old mice were considered young and 8–12-week-old mice were considered adult.