Preliminary Study On Retroviral Vectors For Reversible Immortalization Of Human Hepatocytes

Background and objective: The shortage of hepatocytes material has been an essential problem that restricts the development of bioartificial liver (BAL) and hepatocytes transplantation (HTX). Settlement of the problem will greatly promote the clinic practical value of BAL and HTX. In theory, human hepatocytes (include adult and fetal hepatocytes) are the most ideal. But the source of adult liver donor is very limited and only used in liver transplantation. Further more, adult hepatocytes have poor proliferation potential in vitro. So it is impossible to obtain plentiful adult hepatocytes. Fetal hepatocyte is also difficult to be used widely for ethical reasons. Using animal hepatocytes will take risk of immune reaction and transmission of zoogenic viruses. Hepatocyte strains(such as HepG2,C3A,HHY41,HepZ etc.) have inferior differentiated functions to primary hepatocytes, and most of them are derived from tumor or viral infected primary hepatocytes having problem of security. Reversible immortalization strategies provide new unique means and ideas for cellular materials problems. The rationale is to introduce an immortalizing agent, such as simian virus 40 large T antigen gene(SV40T), into primary cells, expanding the cells in culture, and finally, efficiently removing the immortalizing agent. The resulting cell population will be essentially identical to the starting primary cells, but greatly increased in number and without probability of tumorigenisis and viral infection. Base on this principle, retroviral vectors containing SV40T are constructed in the experiment, the purpose of which is to lay a foundation for reversible immortalization of human hepatocytes. This study consist of three department: the first is "establishment of self-primer bacterial colony polymerase chain reaction (PCR) for screening of loxP-positive recombinant clones", objective of which is to provide effective mothed for rapid identification of vectors to be constructed; the second is "splicing of SV40T gene exons and construction of a retroviral vector pLLTSN ", purpose of which is to delete the intron of SV40T, and shorten the length upported by the National natural Science Foundation of China, No.30100080.of foreign gene DNA benefiting inserting of next genes; and the third is "construction of retroviral vector pLNCTIGlox for reversible immortalization", aim of which is to do preparation for reversible immortalization of hepatocytes.Methods: 1. Synthesized LoxP and vector complementary sequence were used as the upper and lower primer respectively, and bacterial colonies were used directly as the templates of PCR for identification of loxP sequence positive recombinant clones. The positive recombinant clones screened by PCR were evaluated contrastively by restriction endonuclease digestion.2. The two exons of SV40T were amplified respectively by high fidelity PCR using the plasmid pUC19-SV40T as the template.3. The two exons of SV40T gene was spliced by overlapping extension(SOE) and the intron was deleted.4. The retroviral vector pLLTSN was constructed by inserting the spliced 2.1 kb SV40T into the EcoRâ… and BamHâ… sites of the retroviral vector pLXSN.5. The positive recombinant clones were screened and identified by PCR using bacterial colonies directly as templates, and by restriction endonuclease digestion analysis. The spliced 2.1 kb SV40T in pLLTSN was analyzed by DNA sequence and aligned with the BLAST tool on the net. 6. The 2.1 kb SV40T was cut out from pLLTSN and cut in the EcoRâ… and BamHâ… sites of pIRES2-EGFP, resulted vector being named pCTIG. 7. The SV40T-IRES-EGFP DNA fragment from pCTIG into the Xhoâ…  and Notâ…  sites of the retroviral vector pLNCX2, constructing immotalization retroviral vector pLNCTIG.8. The loxP DNA fragment with 4 nt sticky ends same as being cut by Xbaâ… was synthesized and inserted into Xbaâ… site in U3 region in 3'LTR of pLNCTIG.Results:1. LoxP-positive recombinant clones were singled out by self-primer colony PCR. But the result was not shew clear when recombina...