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Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phiC31 integrase in
Zhidong Sun1, Yi Wang, Qiuxia Fu
1Beijing Institute of Transfusion Medicine, Beijing, China.
Background:
The lack of a robust small animal model for hepatitis C virus (HCV) has hindered the development of novel drugs, including internal ribosome entry site (IRES) inhibitors. Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in vivo. This study utilized phiC31 integrase to develop a stable, reproducible and easily accessible HCV IRES mouse model.
Methods:
phiC31 integrase plasmid and the reporter vector, HCV-IRES-luciferase expression cassette (containing an attB site), was codelivered to murine livers using high pressure tail vein injection. HCV IRES-dependent translation reflected by luciferase expression was accurately monitored in vivo by bioluminescence imaging. Genomic integration of the transgene was confirmed by partial hepatectomy and nested PCR. An HCV IRES-targeted short hairpin RNA (shRNA) expression plasmid, sh184, was hydrodynamically transfected into mouse liver to study its inhibition efficacy in vivo.
Results:
phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in mice. The expression of luciferase was stable after 30 days post-transfection and remained so for 300 days only in the livers of mice that were coinjected with the integrase-encoding plasmid. Luciferase levels reduced dramatically after hydrodynamic transfection of sh184.
Conclusions:
These results indicate that this mouse model provides a powerful tool for accurate and long-term evaluation of potential anti-IRES compounds in vivo.
