I recall a graduate student reporting on the characterization of mouse embryonic fibroblast (MEF) lines derived from one of our knockout mice: “Clone 4 does not behave like clone 6.” My response was, “Find me three clones that behave the same.” Such variability was a consequence of the laborious procedures then required to immortalize MEFs. Each clone harboured an inactivating mutation in Tp53, together with multiple additional genetic alterations that differed from clone to clone.

Interestingly, following approximately 20 population doublings at 3% oxygen, MEFs transferred to 20% oxygen neither senesced nor ceased proliferating. Thus, an adaptive process acquired in low-oxygen culture conditions enabled MEFs to overcome the proliferation arrest imposed by atmospheric-level oxygen. Importantly, these immortalized MEFs retained intact p19ARF (encoded by Cdkn2a, also known as Arf) and TP53 function. An application of these findings is that MEF-derived cell lines can now be generated simply by culturing primary MEFs at 3% oxygen for a few weeks. This method is not only far less cumbersome than previous methods, which relied on isolating clones in which Tp53 or Arf were spontaneously or experimentally inactivated, but also yields cell populations whose phenotypes can be more confidently attributed to the original genetic modification introduced into the mouse — no more “clone 4 is different from clone 6.”