Meng FY, Chen ZS, Han M, Hu XP, He XX, Liu Y, He WT, Huang W, Guo H, Zhou P. Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination. World J Gastroenterol 2010; 16(13): 1660-1664 [PMID: 20355246 DOI: 10.3748/wjg.v16.i13.1660]
Corresponding Author of This Article
Ping Zhou, MD, PhD, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical University, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. pzhou57@tjh.tjmu.edu.cn
Article-Type of This Article
Brief Article
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Fan-Ying Meng, Zhi-Shui Chen, Meng Han, Xin-Peng Hu, Yong Liu, Wen-Tao He, Wei Huang, Hui Guo, Ping Zhou, Key Laboratory of Organ Transplantation, Ministry of Education, Ministry of Health, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Xing-Xing He, Institute of Liver Diseases, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Author contributions: Meng FY, Chen ZS and Zhou P designed the study; Meng FY, Han M, Hu XP, He XX, Liu Y and He WT performed the majority of experiments; Guo H and Huang W provided vital reagents and analytical tools; Meng FY wrote the manuscript.
Supported by The Major Scientific and Technological Project of Hubei Province, No. 2007ABD005
Correspondence to: Ping Zhou, MD, PhD, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical University, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. pzhou57@tjh.tjmu.edu.cn
Telephone: +86-27-83662655 Fax: +86-27-83662892
Received: March 9, 2009 Revised: December 20, 2009 Accepted: December 27, 2009 Published online: April 7, 2010
Abstract
AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag).
METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.
RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.
CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
