Engineering Immortalized Bovine Granulosa Cells Using a Triple‐Gene Approach: Mutant CDK4, Cyclin D1, and TERT

ABSTRACT Advancing reproductive technologies in livestock is essential to improve both productivity and genetic potential of cattle. Despite this importance, application of reproductive biotechnologies in cattle breeding remains limited. Bovine granulosa cells (bGCs), which are key components of the ovarian follicle, are critical in female reproduction as they produce steroid hormones and growth factors necessary for oocyte development. However, primary bGCs exhibit restricted proliferative capacity in vitro, limiting their utility in large‐scale studies on mechanisms related to follicular development. To address this limitation, we attempted to immortalize bGCs by co‐expressing human mutant cyclin‐dependent kinase 4 (CDK4 R24C ), cyclin D1, and telomerase reverse transcriptase (TERT) using lentiviral vectors. The resulting immortalized cells (bGCs‐K4DT) displayed extended proliferative lifespans, surpassing 100 population doublings without exhibiting signs of senescence. The transduced cells demonstrated a more active cell cycle profile and higher telomerase activity relative to parental bGCs. Importantly, they retained the bGC‐specific marker, aromatase, albeit at reduced expression levels. This immortalized bGC offers a promising model for investigating the role of bioactive components of platelet‐rich plasma (PRP) in follicular activation and growth, thereby supporting innovations in livestock reproductive technologies.