Generation of Immortalized Per2-Deficient MEFs

Mouse embryonic fibroblasts (MEFs) were obtained from mice without functional Per2 in all body cells^{42 (link)}, or wild-type mice. Briefly, mice containing the ubiquitously expressed Cre recombinase transgene (European Mouse Mutant Archive (EMMA) EM:01149, B6.129-Cre-Deleter: B6.C(129)-Tg(CMV-cre)1Cgn/CgnIbcm) crossed with a conditional Per2 allele^{42 (link)} were backcrossed over ten generations to obtain Per2 homozygous knock-out mice without Cre recombinase. The MEFs subsequently were serially diluted every three days over 30 passages to yield immortalized cells. All lines were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose, stable glutamine, sodium pyruvate and 3.7 g/l NaHCO₃ (PAN Biotech), supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin–streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. Forskolin stimulation (10–100 µM) was used to mimic in vitro the molecular pathway activated by light in mice. Samples were collected at specific time points mentioned in the text.