Application Of Hydrodynamic Gene Delivery In Establishment Of Mouse Model For Monitoring IFN-β Activity And Development Of Tree Shrew Model For HBV Study

Hydrodynamic delivery is a simple and effective non-viral method for intracellular delivery of exogenous substances in whole animals. This procedure employs a pressure generated by a rapid injection of large volume of solution into the vessel to expand the sinusoids and enhance the permeability of membrane for entrance of substance into cell. The gene delivery strategy is gradually popular for study in whole animal, including establishment of animal models, therapy of diseases, DNA immunization, evaluation of drug etc. The substance hydrodynamically delivered is not only DNA, but also protein, oligo nucleotides, genomic DNA and RNA. Based on the technique, we generated two kinds of animal model.Part I Establishment of a mouse model with IFN-βpromoter-driven firefly luciferase reporter in the liver by hydrodynamic gene deliveryType I IFNs, which is represented by IFN-αand IFN-β, play a critical role in the innate immune response. They were initially identified for their antiviral activity and effects on cells of the innate responses. Recognition of the invading virus by immune system and initiation of protein signaling cascades are prerequisites for triggering an effective anti-viral innate immune response. Type I IFNs can be induced by virus infection or by exposure to double-stranded RNA or DNA (dsRNA or dsDNA) in every cell type, and then binds to the cell surface receptors via autocrine or paracrine to trigger the IFN response signaling cascade which results in the expression of interferon-stimulated genes (ISGs). These genes encode a variety of antiviral proteins and inflammatory cytokines, leading to limit the virus spread and protect the host from further viral infection. But even so, most viruses can evade these host immune responses at various degrees. Researches targeting to the mechanism, regulation and application of type I IFN signaling pathways contribute to innate immune network. But few works have been carried out in animals, under a complex immune environment, to evaluate the type I IFN activation. The liver is a crucial site for proteins synthesis as well as metabolism. Many of the immunological components are triggered by signals from hepatocytes. Its function as a major immune organ is gradually appreciated. Many researches are gradually focusing on the effect producted by the innate immunological components in the liver during the infection of hepatitis virus.So, we report a simple, sensitive and visualized method to measure activation of IFN-βin vivo. Mice received only a simple hydrodynamic injection of expression vectors of phage PhiC31o integrase and IFN-βpromoter-driven firefly luciferase reporter. The luciferase expression in mouse liver induced with synthesized dsRNA poly(I:C) can be monitored by bioluminescence imaging, which was consistent with the IFN-βin the sera. The optimal dose of poly(I:C) to induce luciferase expression is 250μg/kg. HCV NS3/4A protease not only inhibited IFN-βproduction, but also blocked the luciferase expression in mouse liver. We clearly demonstrated that our mouse model carried an IFN-βpromoter luciferase reporter gene is a sensitive and stable tool that can be applied to the evaluation of IFN-βpromoter activation under various conditions. The mouse model may not only help further understand the complex biological feature of the type I IFN system in liver, but also screen drug candidates for stimulator or inhibitor of IFN-βgene expression in vivo. It also contributed to the interaction mechanism between the innate immune system and hepatitis virus. Part II Establishment of tree shrews models for HBV study by hydrodynamic gene deliveryHepatitis B virus (HBV) has infected 25% of the world population. 360 million people were chronic infected. Although, the virus is cleared soon for most adults infected, there is a high rate of chronic infection in infants and young children. The mechanism of chronicity of HBV infection is still unclear because of the lack of suitable animal models. HBV has strict host specificity, It only infects some kinds of primates such as human, chimpanzees and tree shrews. However, some primates such as chimpanzee are an endangered species and expensive. Tree shrew lives in tropical and subtropical regions. It is closer to primates than rodents, cats, dogs, rabbits and other animals in biological evolution. Metabolism and anatomical structure are closer to humans than rodents, and easy to be domesticated. Therefore, Tree shrews can be used in many research fields. In recent years, tree shrew model of hepatotropic virus infection have made some progresses. HBsAg positive serum,or purified virus particles were commomly used to inoculate tree shrews for animal model generation. But the rate of infection is low. So we try to generate a tree shrews model of HBV transfection by hydrodynamic with plasmid pAAV/HBV1.2.To establish a method for hydrodynamic-based gene delivery to tree shrews, we explored the conditions of the hydrodynamic transfection. The optimal injection speed is 0.8ml/s and volume is 100ml/kg. And the vectors containing firefly luciferase andβ-galactosidase reporter gene were hydrodynamically injected into tree shrew via the great saphenous vein. The reporter gene was only detected in the liver of tree shrew by bioluminescence imaging andβ-gal staining. The tree shrew was hydrodynamically injected with pHBV1.2 plasmid. At the various times, the sera were collected and in which ALT, HBsAg, anti-HBsAg and HBV DNA were examined by ELISA or real-time fluorescence quantitative PCR respectively. The tree shrews injected hydrodynamically with pHBV1.2 were detected the markers of HBV in their sera. The serum HBsAg and HBV DNA transiently increased, less than a week. Tree shrews treated with cyclophosphamide can not carry HBV longer than those without cyclophosphamide. Hydrodynamic-based injection via the great saphenous vein can be used to deliver the gene to hepatocyte of tree shrews. We preliminary generated a tree shrew model of hepatitis B virus expressing in the liver by the hydrodynamic injection. Our study indicated a new method for establishment of tree shrews models with hepatitis virus infection.Based the technique of hydrodynamic gene delivery, we genearated the mouse model for monitoring IFN-βactivity and preliminary developed the tree shrew model for HBV study.