Abstract
The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.