Immortalized ERCC1 MEF Cell Assay

Timed matings were performed between Ercc1 heterozygous mice and pregnant females were culled by cervical dislocation at E12.5. Primary MEF cultures were obtained and immortalized using the SV40 large T antigen as described previously^{23 (link)}. Briefly, pBABE-SV40-Puro virus was produced using Plat-E cells (Cell Biolabs). 48 hours following transfection, the culture medium containing retroviruses was collected and passed through a 0.22 μm filter. The filtered retrovirus was mixed 1:1 with complete medium and supplemented with 4 μg.ml^-1 polybrene, the resulting infective medium was added to primary MEF cultures. Transformed clones were selected for 10 days using 3.5 μg.ml^-1 puromycin. Sensitivity to DNA-damaging agents was determined by seeding 500 transformed MEFs per well of a 96-well flat-bottom plate and exposed to mitomycin c (MMC) or ultraviolet (UV) irradiation. After 10 days of culture post-exposure the MTS cell viability reagent (Promega) was added and plates incubated at 37 °C for 4 hours, and absorbance at 492 nm was measured.