Abstract
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 inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. 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 broadly to immortalize other mouse primary cell types. Key features • CRISPR-based knockout of the Tp53 gene enables efficient immortalization of mouse embryonic fibroblasts (MEFs) in under three weeks. • Immortalization requires a Neon electroporator or a comparable system to transfect cells with the Tp53 CRISPR constructs.