Establishment of an immortalized sheep mammary epithelial cell line for studying milk fat and protein synthesis

Abstract The mammary gland, crucial for milk production in mammals, presents challenges for in vitro study due to its complex structure and limited cell lifespan. We addressed this by introducing the SV40 large T antigen into primary mammary epithelial cells (MECs) from sheep, creating an immortalized T‐tag MEC line. This line, stable for over 50 passages, maintained typical epithelial cell morphology during long‐term culture. Through transcriptome sequencing and validation, we discovered 3833 differentially expressed genes between MECs and T‐tag MEC line, encompassing key biological processes and signaling pathways like cell cycle, p53, and cancer. The cell line, expressing MEC markers (KRT8, KRT18, proliferating cell nuclear antigen, SV40, CSN2, and acetyl‐CoA carboxylase alpha), proved capable of synthesizing milk fat and protein. Despite its infinite proliferation potential, the T‐tag MEC line showed no tumor formation in mice or cell migration in vitro, indicating stability. This development offers a valuable resource for studying MECs in dairy sheep, facilitating the advancement of long‐term culture systems and in vitro lactation bioreactors.