Establishment Of A Mouse Model With Long-term Expression Of HCV Full-length Genome

Hepatitis C virus (HCV) infection is a global public health problem,75%-85% of acute infected patients will develop chronic hepatitis, among which 10%-20% cases can lead to liver cirrhosis and hepatocellular carcinoma. At present, the chronicity mechanism after HCV infection is thought due to the disorders of immune functions ,and damage of hepatocytes is related with infiltration of immunocytes and cell factors in the liver . Nevertheless, the researches about the pathogenic mechanism of HCV were hampered by a lack of convenient small animal models.Therefore, searching for the valued HCV full-length genome expression models for hepatitis C prevention and treatment is very desirable.We adapted hydrodynamic-base procedure and phage integrase system for the establishment of a mouse model for long-term expression of full-length HCV genome. It can be used to evaluate the effect of HCV long-term expression on immune system and hepatic injury. The main contents and the results of our research will be discussed below:1.Construction and identification of eukaryotic expression plasmid (pCI-attB- AerApoEAATP-Fluc-EMCV IRES-HCV )The eukaryotic expression vector pCI-attB-AerApoEAATP contain the liver- specific promoter AerApoEAAT(AAA) and attB site that can be attached by phage integrase , and the activity of reportor gene Fluc can reflect HCV expression level. By connected gene Fluc,EMCV IRES and HCV in order after the promoter hAAT, we constructed the recombinant plasmids pCI-attB-AerApoEAATP-Fluc- EMCV IRES- HCV .2.Establishment of a mouse model with short-term expression of HCV full-length genome.Hydrodynamics-based transfection,involving a large-volume(8%-12% of mouse weight)and high-speed(4-8s)intravenous injection of naked DNA via tail vein,has been thought as a simple,convenient, stable,efficient and safe gene delivery method in vivo. the plasmid pCI-attB-AerApoEAATP-Fluc-EMCV IRES-HCV was transfected into mice by hydrodynamics-based procedure and the bioluminescence imaging was used to real time quantitatively detect the dynamics of Fluc expression in mice livers. Subsequently, expression of the HCV full-length genome were further confirmed by RT-PCR, Western-blotting and the immunity histochemical method. However, this model has obvious deficiencies in researching the mechanisms of chronic viral hepatitis and evaluation of antiviral drugs. It is necessary to establish a long-term expression model.3. Establishment of a mouse model with long-term expression of HCV full-length genome.It is reported that phageφC31 integrase is a site-special recombinase that can catalyses the recombination reaction between the attB recognition site in an episomal plasmid and pseudo-attP site(attP) in the mouse chromosomes.Thus, we combined the hydrodynamics andφC31 integrase to get the long-term transgene expresstion in mouse liver.In this study, the plasmid pCMV-int was cotransfected with plasmid pCI-attB-AerApoEAATP-Fluc-EMCV IRES-HCV or pCI-attB-AerApoEAATP-Fluc (control) into mice by hydrodynamics-based procedure and the bioluminescence imaging was used to real time quantitatively detect the dynamics of Fluc expression in mice livers. Study identified that HCV got a long-term expression in mice cotransfected with pCMV-int, of which the Fluc and HCV genome kept stable expression and lasted for over 90 days.Mice from two groups of a long-term expression for Fluc and HCV were operated a surgery of the two-thirds partial hepatectomy(PH), Fluc expression in pCI-attB-AerApoEAATP-Fluc-EMCV IRES– HCV group didn't recovered after 14 d past PH with livers regeneration while opposite result was obtained in in control group.But nested-PCR identified the integration of the HCV expression frame into mice chromosomal DNA at??pseudo-attP''sites. Sequence analysis confirmed the hot site was mpsL1 located at mice chromosomal 2 generating two recombination junction sites (attR and attL), and there were 1-8 bp micro deletion near the core area TG.These results indicated that HCV full-long genome was indeed conformed in the mouse genome and its expression in vivo might suppressed liver regeneration. In summary, combined hydrodynamic transfection with phage integrase system , we established a mouse model with long-term expression of HCV full-length genome. This model will contribute to the pathogenic researches of HCV,the screening of HCV vaccine and the evaluation of anti-HCV drugs in vivo. However,how to improve the expression level and its stability of the exogenous gene in mice is still important issues to solve.