Control Of HCV NS5B Protein Expression In Transgenic Mice

Currently, there are about 170 million people worldwide infected with the hepatitis C virus (HCV).HCV infection often leads to severe chronic liver disease including fatty liver,cirrhosis and liver cancer,and does seriously harm to human beings'health.So far,there has not been any vaccine to prevent HCV infection yet,and the antiviral therapies are of high cost-effectiveness,side effects and limited efficacy.Therefore,the hepatitis C has gained great attention form all over the world as the hepatitis B did.It has been a long time since scientists committed to doing researches on the prevention and treatment of HCV infection,but this progress advanced slowly.The high variability of HCV contributed to the slow advacncement a lot,and lack of stable and effective animal models for HCV infection also made a serious impedient to the study of the pathogenesis mechnism, antiviral drugs screening and effective vaccines development.At present,animal models for HCV infection mainly include HCV-infection models,human-liver-chimeric mouse models and transgenic mouse models. However, almost all these types have its intrinsic defects respectively.HCV NS5B protein,an RNA-dependent RNA polymerase(RdRp),plays a key role in the process of HCV replication.Given the fact that the crystal structure and the function of NS5B has been known, the NS5B protein is an ideal target for the development of anti-HCV pharmacy.This study aims to establish a transgenic mouse model which could liver-specifically and conditionally expresses HCV NS5B protein by means of the joint use of Tet-on regulatory system and Cre/LoxP gene-knockout system.Research contents and results are as follows.1.Construction of eukaryotic vector expressing the HCV NS5B protein under dual control of the Tet-on and Cre/LoxP system.By utilizing the eukaryotic expression vector pBI-3 as vector framework,the luc gene,the BGH PolyA tail and the ns5b gene fragments were orderly inserted into the downstream of the promoter,and a LoxP site was introduced respectively in the upstream of the luc gene and downstream of the BGH PolyA tail.The eukaryotic expression vector pBI-3/luc-BGH PolyA -NS5B,which could be dually-controlled by Tet-on and Cre/loxP systems,were successfully constructed.The recombinant vector contains regulatory elements of Tet-on and Cre/loxP systems,together with dual-reporter gene fragments.Such designs could not only avoid undetected expression stringently,but also provides a simple,sensitive and accurate detection method in latter experiments for the screening of transgenic mouse with low background expression and strong inducibility.The successful construction of PBI-3/luc-BGH PolyA-NS5B vector laid a good foundation for the establishment of transgenic mice which could strictly and conditionally regulate the expression of HCV NS5B protein.2.Establishment of triple transgenic mice conditionally expressing HCV NS5B protein under Dox induction.The second part of the study is to preparae the triple transgenic mice which can conditionally and liver-specificlly express HCV NS5B protein under the dual-control of Tet-on regulatory system and Cre/LoxP gene knockout system.The recombinant vector was linearized and sent to Shanghai Research Center for Model Organisms to prepare NS5B transgenic founder mouse.Six NS5B transgenic founder mouse were gained and identified using PCR and Southern bolt, and then hybrided with C57BL/6 mice to obtain the first offsprings(F1),respectively.NS5B-transgene positive offspring mouse were screened using PCR.A part of the F1 transgene generations were mated with the C57BL/6 mouse to breed expand generations,and the offsprings were screened using PCR to detect the genetic stability of foreign transgene.Another part of the F1 transgenes were hybrided with rtTALAP-1/LC-1 double transgenic mouse liver-specificlly expressing rtTA protein and Dox-conditionally expressing Cre recombinase.Offsprings were called tri-transgenic mouse and were screened using PCR analysis of Lap transgenic fragment,Cre transgenic fragment,and ns5b transgenic fragment.The triple fragments positive transgenic mouse were fed with Dox(doxycycline hydrochloride) drinking water a week . These tri-transgenic mouse were injected intraperitoneally with D-Luciferin and detected whether reporter gene were effectively exprssed in the liver tissue using small animal in vivo optical imaging system(BLI).Subsequently,the expression of Cre recombinase and the NS5B protein in liver and other tissues of these imaging-positive mouse were analyzed through immunohistochemistry.The results showed that the light signal was only clearly detected in the liver of these tri-transgenic mouse induced with Dox,indicating that the regulation of the tri- transgenic mouse and the specificity are good.Immunohistochemistry staining confirmed that Cre recombinase specifically localized in the nucleus of hepatocyte,NS5B protein specifically distributed in the cytoplasm hepatocyte.In summary,tri-transgenic mouse model conditionally and liver-specificlly express HCV NS5B protein were established by combining Tet-on regulatory system and Cre/LoxP gene knockout system.Expressed NS5B protein in this model mice was liver targeted and was of strong inductivity,which provided a good experimental basis for later evaluation of the stability and effectiveness of tri-transgenic mouse.