三种丙型肝炎病毒转基因小鼠系的同时制备

为研究丙型肝炎病毒的致病致瘤机理及结构基因与非结构基因3区(NS3)的功能及其在HCV感染致病中的作用,建立一个HCV分子治疗的动物模型,构建了含金属硫蛋白启动子和HCV结构基因或NS3基因的质粒,将两者等量混合后用显微注射法接种于昆明白小鼠受精卵内制备转基因小鼠.通过PCR筛选获得三种整合HCV结构基因或/和NS3基因的首建鼠.结果表明:a.注射后卵存活率与仔鼠出生率分别为81%、30%;b.检测60只G0代小鼠,结构基因整合鼠6只(10%),NS3基因整合鼠4只(6.7%),双基因整合鼠9只(15%),总整合率为31.7%;c.RT-PCR法检测阳性鼠肝中有靶基因mRNA的转录;d.4只首建鼠与正常鼠回交获得38只G1小鼠,其中20只为整合鼠,整合率为52.6%;e.转基因鼠表型迄今无明显异常.表明一次显微注射同时获得了三种整合HCV结构基因或/和NS3基因的转基因小鼠.

Production of Three Transgenic Mouse Lineages Which Simultaneously Carries Structural Gene and/or Non-structural Gene 3(NS3) Region of Hepatitis C Virus

Hepatitis C virus(HCV) is a major causative agent of non-A non-B chronic hepatitis and also associated with the development of hepatocellular carcinoma. To understand the mechanism of pathogenesis and tumorigenesis of HCV and the role of the structural protein and NS3 protein in the HCV infection,three lines of transgenic mice carrying the HCV structural gene or/and NS3 gene, in which these genes are expressed under the control of the mouse metallothionein promoter, were produced simultaneously. These animals were produced by microinjecting into the pronuclei of fertilized mouse eggs with the mixture of construct carrying the structural gene and NS3 gene of HCV. The results were as follow: 1) The zygote's survival rate and birth rate were 81% and 30%, respectively; 2) After screening 60 mice that developed from the microinjected eggs by PCR, 6(10%)mice carrying the structural gene, 4(6.7%)mice carrying NS3 gene and 9(15%) carrying double genes.The total integrate rate was 31.7%; 3)The expression of transgene in the liver of Tg mice was analyzed by detection of the mRNA using RT-PCR; 4) 38 G1 mice were obtained by crossbred founder mice and normal mice, 20 mice were integrated(52.6%) with transgene; 5) No phenotypic abnormalities have been seen to date. The model may create the opportunity to examine the role of the structural and NS3 protein in HCV pathogenesis and tumorigenesis, and to assess the antiviral potential of the pharmacological agents and vaccine.