Immortalized Mouse Embryonic Fibroblasts for Genetic Analyses

Primary MEFs were generated from E14.5 embryos by standard methods. The LARP4 KO MEFs were described (Mattijssen et al., 2017 (link)). Each MEF cell line was derived from a different embryo, all females. The MEFs used to generate the data in Figure 3 were immortalized as described using SV40 Large-T antigen (Mattijssen et al., 2017 (link)), the experiment in Figure 2 was performed using three independent WT- and 3 LARP4 KO MEF cell lines (N = 3, biological replicates). MEFs used for the IFNα ActD experiment in Figure 5 were cultured following the 3T3 protocol for spontaneous immortalization (Todaro and Green, 1963 (link)). The experiment in Figure 5 was performed using two independent WT- and 2 LARP4 KO MEF cell lines (N = 2, biological replicates). The immortalized MEFs and HEK293 cells were cultured in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals). HEK293 cells are not commonly misidentified. Nonetheless DNA from these cells was authenticated by ATCC via STR (short tandem repeat) profiling. Standardized testing (ATCC) had verified that the cells were free of mycoplasma infection.