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.
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.
Authors: N Kobayashi; T Fujiwara; K A Westerman; Y Inoue; M Sakaguchi; H Noguchi; M Miyazaki; J Cai; N Tanaka; I J Fox; P Leboulch Journal: Science Date: 2000-02-18 Impact factor: 47.728
Authors: Umberto Baccarani; Andrea Sanna; Alessio Cariani; Mauricio Sainz-Barriga; Gian Luigi Adani; Anna Maria Zambito; Giuseppe Piccolo; Andrea Risaliti; Alessandro Nanni-Costa; Lorenza Ridolfi; Mario Scalamogna; Fabrizio Bresadola; Annibale Donini Journal: Liver Transpl Date: 2003-05 Impact factor: 5.799
Authors: Kyle M Schachtschneider; Regina M Schwind; Kwame A Darfour-Oduro; Arun K De; Lauretta A Rund; Kuldeep Singh; Daniel R Principe; Grace Guzman; Charles E Ray; Howard Ozer; Ron C Gaba; Lawrence B Schook Journal: Oncotarget Date: 2017-06-29