Our goal was to establish in vitro models using minipig brain cells to Establish the Cell Lines Detecting Circadian Rhythm Gene Expression. We isolated various brain cell types and generated continuously proliferative cell lines from primary cultured cells. These cell lines were extensively characterized using relevant markers to ensure suitability for studying circadian rhythm-related genes. Through CRISPR-Cas9 technology, we engineered cell lines expressing key circadian rhythm-related genes like PER, REV-ERBα, and HTR1A, confirmed by red fluorescent markers. Subsequent RNA sequencing analysis revealed a network of ten genes crucial to circadian function within these engineered cell lines. This comprehensive characterization not only provides foundational insights into circadian rhythm mechanisms but also offers potential avenues for future research and therapeutic interventions targeting circadian disruptions across the lifespan. Our findings significantly contribute to the burgeoning field of chronobiology, providing a robust in vitro model for studying circadian rhythm regulation. By deepening our understanding of circadian regulation in health and disease, our study underscores the importance of incorporating cellular models into circadian biology research and highlights the translational potential in improving human health and longevity.