TRANSGENIC MOUSE MODEL FOR STUDY OF HEPATITIS C

虽然丙型肝炎病毒（HCV）是世界范围内发病率和死亡率的主要原因，但病毒基因表达对感染细胞的影响仍不清楚。 在此之前，我们报道了表达HCV结构蛋白（核心，E1和E2）的转基因小鼠的构建，并表明HCV结构蛋白的表达在这种动物模型中不直接致细胞病变。 使用DNA免疫，我们能够诱导抗体，T细胞增殖和细胞毒性T细胞反应对HCV核心，但只有对包膜蛋白在这些转基因动物的抗体。相比之下，细胞免疫应答似乎主要针对野生型小鼠中的包膜蛋白。 这些结果表明，在野生型小鼠中针对HCV结构蛋白的CTL应答的层次（包膜蛋白>核心蛋白），以及在转基因小鼠中在T细胞水平上对核心蛋白的免疫忽略而对包膜蛋白的免疫耐受。 本实验室还建立了表达HCV全长多聚蛋白的转基因小鼠，并正在开发HCV转基因的诱导表达系统。基于四环素调控系统，我们建立了一个双元转基因模型，其中两个转基因，SV40 T抗原（TAg）和LacZ，可以通过施用四环素在肝脏中严格调控的条件表达。 使用小鼠白蛋白或小鼠尿蛋白（MUP）启动子在一组转基因小鼠中实现四环素应答性转录激活因子（tTA）的肝脏特异性表达。 将这些小鼠与在tTA调节的启动子控制下携带TAg或LacZ的转基因小鼠杂交。对tTA和TAg（或LacZ）转基因小鼠的分析表明，转基因的肝脏特异性表达可以被抑制到不可检测的水平，并通过四环素给药和停药以可逆的方式进行调节。tTA和TAg转基因小鼠发展为肝细胞腺瘤和增生，可通过持续四环素给药预防。该实验证明了该二元转基因模型在以肝脏特异性方式研究任何潜在的感兴趣基因的生理功能中的价值。我们目前正在评估使用该模型系统的HCV蛋白在肝脏中的表达调控。这些动物将提供一个有用的动物模型，不仅解决问题的免疫发病机制和细胞病变的潜在的HCV基因产物，但也研究HCV在体内的复制。

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Previously, we reported the construction of transgenic mice expressing HCV structural proteins (core, E1 and E2) and showed that expression of HCV structural proteins is not directly cytopathic in this animal model. Using DNA immunization, we were able to induce antibodies, T cell proliferative and cytotoxic T cell responses against the HCV core but only antibodies against the envelope protein in these transgenic animals. In contrast, the cellular immune responses appeared to target predominantly against the envelope proteins in wild-type mice. These results suggest a hierarchy of CTL response against the HCV structural proteins (envelope>core) in the wild-type mice, and an immunological ignorance toward the core but tolerance toward the envelope proteins at the T cell level in the transgenic mice. Our laboratory has also generated transgenic mice expressing HCV full-length polyprotein and is developing a system for the inducible expression of HCV transgene Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mouse albumin or mouse urinary protein (MUP) promoter was used to achieve liver-specific expression of the tetracycline-responsive transcriptional activator (tTA) in one set of transgenic mice. These mice were crossed with transgenic mice carrying either TAg or LacZ under the control of tTA-regulated promoter. Analyses of mice transgenic for both tTA and TAg (or LacZ) revealed that the liver-specific expression of the transgenes could be suppressed to undetectable level and regulated in a reversible fashion by tetracycline administration and withdrawal. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. This experiment demonstrates the value of this binary transgenic model in studying the physiological functions of any potential genes of interest in a liver-specific manner. We are currently evaluating the regulated expression of HCV proteins in the liver using this model system. These animals will provide a useful animal model not only to address issues of immunopathogenesis and cytopathic potential of HCV gene products but also to study HCV replication in vivo.