Establishment Of A Novel Transgenic Mouse Model For Visually Evaluating The Activity Of HCV RdRp In Vivo

?Background? Hepatitis C virus(HCV) belongs to the genus Hepacivirus which is a member of the Flaviviridae family, which is accounted for human chronic hepatitis, cirrhosis, and hepatocellular carcinoma(HCC). HCV infection is a global public health problem with approximately 170 million individuals infected worldwide. However, there is no vaccine for HCV. HCV is a positive single-stranded RNA virus. The viral genome is approximately 9,600 nucleotides including 5'-untranslated region(5' UTR), an open reading frame(ORF) and 3'-untranslated region(3' UTR). 5' UTR and 3' UTR are the important regulatory elements for RNA replication and translation. ORF encodes a large polyprotein of about 3,000 amino acids. The polyprotein precursor could be cleaved by the host and viral proteases into 10 mature viral proteins including three structural proteins(core, E1, E2) and seven nonstructural proteins(p7, NS2, NS3, NS4 A, NS4 B, NS5 A, NS5B). Previous research reported that NS3, NS4 A, NS4 B, NS5 A and NS5 B were involved in regulation of viral RNA replication. Especially, NS5 B is not only a key RNA-dependent RNA polymerase(Rd Rp) during viral RNA replication, but also expected as a potential target for anti-HCV therapy. Currently, Sofosbuvir which specific target NS5 B has already been approved by FDA because of its clinically curative effect. It confirms that NS5 B is an ideal target for anti-HCV treatment. However, it is difficult to be widely used for the high price. The main reason for the slowly development of NS5 B inhibitors is lack of the experimental evaluation system. Taken advantage of the further understanding of HCV RNA replication and the reverse genetic manipulation technology, a new HCV minigenome system has been constructed containing HCV 5' UTR, 3' UTR and a reporter gene. Cellular experimental results indicated that the system could be a model of HCV RNA replication in vitro, therein it might be an efficient tool for evaluation the activity of Rd Rp. Nonetheless, it couldn't be a real-time indicator as it is in vitro. Therefore, it is necessary to establish an animal model which could be used to evaluate the activity of HCV Rd Rp in vivo. It could also be beneficial for HCV inhibitor development.?Objective? To generate a novel transgenic mouse model for visually evaluating the activity of HCV Rd Rp using hydrodynamics-based gene delivery via retro-orbital sinus transfection.?Methods? HCV NS3-5B transgenic founder mice were generated by microinjection of the linearized vector of p IRES2-EGFP-NS3-5B into mouse fertilized eggs, which were identified by PCR. The offspring could be got by crossing NS3-5B transgenic founder mice and C57BL/6 mice. The next generations were identified by PCR, RT-PCR and Western blot. Continuous cross breeding and screening were performed until the genetic stability of NS3-5B transgenic mice were selected out. The serum was separated from eyeball blood. The ALT and AST in the serum of C57BL/6 mice and NS3-5B transgenic mice were compared. HE staining of liver, kidneyand spleen was performed to detect the histomorphological changes in transgenetic mice. According to the gene sequence of HCV minigenome and HDV nucleic acid enzyme, specific primers were designed for amplifying A and B segments from pc DNA3.1(+)-(-)3UIRr G(-)5. The segments of A and B were amplified respectively by overlap PCR and were then digested and cloned into pc DNA3.1(+)-MCS, which construct the recombinant plasmid of pc DNA3.1(+)-H0299 G. It was confirmed by PCR, enzyme digestion and sequencing. NS3-5B transgenic mice were transfected by pc DNA3.1(+)-H0299 G by HGD via retro-orbital sinus. The expression of GLuc was detected by Western blot, in vivo imaging and immunohistochemistry on the tissues. In addition, the liver damage in transgenetic mice was indicated by the changes of ALT, AST and structure in the liver tissue. Furthermore, the expression of GLuc in serum and the luminous intensity in NS3-5B transgenic mice was tested using the principle of chemiluminescence.?Results? The enzyme-digested products of p IRES2-EGFP-NS3-5B were seperated via electrophoresis and acquired the NS3-5B transgenic carrier segment the size of which is in accord with expectation. PCR amplification by which NS3-5B transgenic founder mice were identified obtained the expected size of gene segment. The progeny was produced by crossing mate of HCV NS3-5B transgenic founder mice mated with C57BL/6 mice. PCR, RT-PCR and Western blot were investigated to screen progeny mice and those results showed that the transgenetic model mice strain with genetic stability has been established. The ALT and AST in serum and histomorphological analysis on liver tissue of NS3-5B transgenic mice were indicated that there was no change compared with those in C57BL/6 mice. Western blot, in vivo imaging of single tissues, and immunohistochemical staining revealed that the liver could be specifically transfected by HGD via retro-orbital sinus. The detection of the ALT and AST in serum and HE staining of liver tissue suggested that through the technology can cause slight hepatic injure, it could be self-repaired without physiological functional changes.After the plasmid of pc DNA3.1(+)-H0299 G was transfected in vivo, real-time evaluation of the GLuc expression in serum and in vivo bioluminescent imaging dynamically in NS3-5B transgenic mice showed that the GLuc expression was highly significant in NS3-5B transgenic mice compared with those in C57BL/6 mice. It indicated that HCV minigenome has been successfully expressed in transgenetic modified mice.?Conclusion? In this study, NS3-5B transgenic mouse model with the expression of HCV replication complex was successfully established. After the pc DNA3.1(+)-H0299 G was transfected into NS3-5B transgenic mice by HGD via retro-orbital sinus, the GLuc expression confirmed that HCV minigenome could be expressed under the regulation of HCV Rd Rp in living mice. The novel transgenic mouse model could be used to visually evaluatethe activity of HCV Rd Rp and as an experimental platform for evaluating and screening of HCV NS5 B inhibitors.