ERas-Null Mice Generated Directly from Embryonic Stem Cells in a Lipid-rich Medium Enable the Discovery of A Novel Role in Craniofacial Development

Summary Generating mice entirely from embryonic stem cells (ESCs) via tetraploid complementation, termed all-ESC mice, is considered the gold standard for pluripotency testing. In this study, we examined the effects of our newly reported lipid-rich 2i/LIF medium (2iLA) on ESCs for genetic engineering. As a proof of principle, we targeted the ERas gene using the 2iLA medium, since ERas -null mice displayed no noticeable phenotype in a previously developed mouse model through chimeras. We demonstrated that the 2iLA medium effectively supports the creation of both male and female ERas -null all-ESC mice, emphasizing the advantages of this culture medium. Unexpectedly, we observed that ERas -null all-ESC mice produced with the 2iLA medium exhibited a non-Mendelian lethal craniofacial anomaly, which can be mitigated by using the lipid-free 2i/LIF medium. Our findings not only highlight the potential of the 2iLA medium for gene targeting but also reveal a novel lipid-associated role of the ERas gene in craniofacial development. Our system offers a unique alternative for studying developmental gene functions unachievable with traditional methods and provides a novel platform for the rapid construction of mouse models. Graphic Abstract All-ESC pups from wild-type (WT) ESCs cultured in lipid-rich 2iLA medium are normal. In contrast, both male and female ERas -null isogenic all-ESC pups produced from ESCs in 2iLA medium display a non-Mendelian craniofacial anomaly. This phenotype is alleviated when the ESCs are grown in a lipid-free 2iL medium.