Immortalization of a Primary Bovine Mammary Epithelial Cell Line by the SV40 Large T-antigen Gene

Abstract: In this study, we report an immortalized cell line expression of SV40 large T-antigen established from a Chinese Holstein primary mammary epithelial cell (CMECs) cultured in vitro. The plasmid that carried the SV40 large T-antigen sequence was introduced into mammary epithelial cells by retroviral mediation. Following injection, cells were cultured under puromycin for selection and 10% resistant cells remained after two weeks. Four immortalized bovine mammary epithelial cell colonies were obtained, but only a single colony was surpassed over 50-passage and was designated CMEC-H. The obtained clone was characterized with respect to their morphogenetic behavior, long-term proliferative potential, and differentiation characteristics. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical “cobblestone” morphology characteristic with obvious boundary that were more homogeneous than in the primary mammary epithelial cells. And the homogenously polygonal of immortalized cell was maintained from passage 1 to 50. The population growth rate between immortalized cells at passage 5 or 50, doubled in number within 36 h, was not significantly different but was faster than in the primary cells. The typical and representative karyotype of CMEC-H was counted at 100 chromosomes, which was more than the normal diploid chromosome number 60. The telomerase expression of CMEC-H had consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line failed to induce tumor formation in nude mice. The immortalized epithelial cell expressed epithelial cell markers, including cytokeratins CK7, CK8, CK18, and CK19. The gene and protein expressions of caseins (α_S1-casein, β-casein, and κ-casein) indicated that the immortalized CMEC-H maintained the milk protein synthesis function of epithelial cells. We conclude that CMEC-H may become a valuable reagent for studying the secretion mechanism of mammary gland.

Keywords: bovine mammary epithelial cell; immortalization; SV-40 large T-antigen;