A new method to immortalize primary cultured rat hepatocytes

In conventional methods of establishing hepatocyte cell lines, the immortalizing gene alone is introduced into hepatocytes. We designed a new method in which not only the immortalizing gene, the simian virus-40 large T-antigen (SV-40 Tag) gene, but also a drug-resistant gene, under the control of an albumin enhancer/promoter, were introduced into hepatocytes to efficiently obtain immortalized hepatocyte cell lines.

The plasmid pAPUR contains the puromycin-resistant gene under the control of an albumin enhancer/promoter, and the pSVTag contains the early region of SV-40 enhancer/promoter and the SV-40 Tag gene. Both pAPUR and pSVTag were transferred into isolated rat hepatocytes by electroporation. After these cells were cultured on a collagen-coated dish for 24 hours, puromycin selection was started. Expression levels of albumin, alpha-fetoprotein (AFP), SV-40 Tag, and cytokeratin 19 (CK 19) in the transformed cells were evaluated by western analysis, immunocytochemical staining, and RT PCR.

Approximately 3 weeks after transfection, five or six colonies appeared on the dish. Twenty strains were obtained by cloning these cells. All strains that were similar to immature hepatocytes expressed albumin and SV-40 Tag, although CK 19 was not detected. AFP expression was detected in 33% of these strains.

All clones cotransfected by pAPUR and pSVTag expressed albumin. Our new method may be useful to establish hepatocyte cell lines.