title: Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Fan Ying
authors.initials: FY
authors.lastname: Meng
authors.forename: Zhi Shui
authors.initials: ZS
authors.lastname: Chen
authors.forename: Meng
authors.initials: M
authors.lastname: Han
authors.forename: Xin Peng
authors.initials: XP
authors.lastname: Hu
authors.forename: Xing Xing
authors.initials: XX
authors.lastname: He
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Wen Tao
authors.initials: WT
authors.lastname: He
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Hui
authors.initials: H
authors.lastname: Guo
authors.forename: Ping
authors.initials: P
authors.lastname: Zhou
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000473:pathology; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics*; D013552:Swine; D013556:Swine, Miniature
chemical_list: D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A