An efficient method for immortalizing mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inefficient or alter the physiological properties of the cells. Here, we describe an optimized protocol for immortalizing MEFs via CRISPR-mediated deletion of the Tp53 gene. This method is highly efficient and consistently generates immortalized MEFs, or iMEFs, within 14 days. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted to immortalize other mouse primary cell types. Key FeaturesO_LICRISPR-based knockout of the Tp53 gene enables efficient immortalization of mouse embryonic fibroblasts (MEFs) in under 2 weeks. C_LIO_LIImmortalization requires a Neon electroporator or another comparable electroporation system to transfect cells with the Tp53 CRISPR constructs. C_LI