Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phiC31 integrase in mouse model.
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.
Sun
Zhidong
Z
Beijing Institute of Transfusion Medicine, Beijing, China.
Wang
Yi
Y
Fu
Qiuxia
Q
Zhou
Yong
Y
Jia
Shuaizheng
S
Du
Juan
J
Peng
Jianchun
J
Wang
Yingli
Y
Yang
Shuhua
S
Zhan
Linsheng
L
D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D005814:Genes, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006526:Hepatitis C / Q000188:drug therapy*; D019426:Integrases / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice / Q000235:genetics*; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D010442:Peptide Chain Initiation, Translational; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism; D014162:Transfection
D000998:Antiviral Agents; D008156:Luciferases; D019426:Integrases