Immortalization of Primary Mouse Embryonic Fibroblasts

All cell lines were maintained in standard growth medium -DMEM/high glucose, 1 mM glutamine, 10% FCS (20% for MEFs), 1x Penicillin/Streptomycin. Transient transfections were performed using polyethyleneimine (PEI) reagent as described previously24 (link). Sept7 floxed mouse (Sept7^tm1Mgl)14 (link) embryonic fibroblasts were generated from E14 day embryos. Aseptically minced embryos after removing head and red organs were further disaggregated by mincing in a solution of Trypsin-EDTA/DNAseI and incubation with 5 mm glass beads with vigorous shaking at RT for 15 min. The isolated cells were transferred to a fresh tube and resuspended in standard growth medium with 50 μg/ml gentamycin, plated in T25 flasks and propagated at 37 °C with 5% CO₂. The cells were splitted 1:4 and maintained in the same growth medium. To immortalize primary MEFs, cells were co-transfected with pSV40Tag encoding simian virus 40 large T-antigen and pREP8 plasmid (Invitrogen) in a 10:1 mixture; colonies were selected with 2 mM histidinol (Sigma). For proliferation assays, MEF clones were seeded in 96 well plates and on alternate days viable cell population were quantified by WST-1 assay (Roche).