Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination.
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.
Meng
Fan Ying
FY
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.
Chen
Zhi Shui
ZS
Han
Meng
M
Hu
Xin Peng
XP
He
Xing Xing
XX
Liu
Yong
Y
He
Wen Tao
WT
Huang
Wei
W
Guo
Hui
H
Zhou
Ping
P
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
D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A