TRANSGENIC MOUSE MODEL FOR STUDY OF HEPATITIS C

尽管丙型肝炎病毒（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体内影响尚不清楚。在此之前，我们报道了表达HCV结构蛋白（core， E1和E2）的转基因小鼠的构建，并表明HCV结构蛋白的表达在该动物模型中不直接引起细胞病变。使用DNA免疫，我们能够在这些转基因动物中诱导针对HCV结构蛋白的抗体和T细胞增殖反应，但不能诱导细胞毒性T细胞反应，这表明T细胞水平的耐受性更难以打破。我们的实验室也在培育表达HCV全长多蛋白的转基因小鼠，并开发了一种使用四环素诱导系统诱导表达HCV转基因的系统。使用这种方法，转基因可以以时间和组织特异性的方式表达，也可以避免在胚胎发育过程中发生的胸腺选择过程。这对于研究对外来抗原（如病毒蛋白）的免疫应答具有很大的优势，因为表达病毒基因的转基因小鼠通常对病毒基因产物具有耐受性。我们开发了两种转基因小鼠模型，每种模型都有条件地表达SV-40 T抗原（Tag）或LacZ。为了获得这些动物，我们首先在肝脏特异性白蛋白启动子（AlbtTA）或小鼠尿蛋白启动子（MuptTA）的控制下产生表达tTA反激活子的转基因小鼠，然后将这些小鼠与含有tetTag或tetLacZ转基因的小鼠杂交。对后代进行基因分型，并皮下植入缓释四环素微丸进行四环素治疗。在Tc植入21天后分析转基因基因的表达情况。在AlbtTA/tetTag或MuptTA/tetTag双阳性的动物肝脏中检测到标签表达。在这两种情况下，转基因表达仅局限于肝脏，并且可以被Tc治疗完全消除，RT-PCR显示没有转基因转录。退出Tc后，标签蛋白合成恢复。同样，MuptTA/tetLacZ双阳性小鼠的LacZ表达受到Tc处理的严格调节。我们目前正在使用该模型系统评估HCV蛋白在肝脏中的调控表达。这些动物将提供一个有用的动物模型，不仅可以解决HCV基因产物的免疫发病机制和细胞病变潜力问题，还可以研究HCV在体内的复制。-细胞病变效应/诱导表达/免疫反应/动物模型

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 antibody and T cell proliferative responses but not cytotoxic T cell response against HCV structural proteins in these transgenic animals, suggesting that tolerance at the T cell level is more difficult to break. Our laboratory is also generating transgenic mice expressing HCV full-length polyprotein and has developed a system for inducible expression of HCV transgene using the tetracycline-inducible system. Using this method, transgenes could be expressed in a time- and tissue-specific manner and it is also possible to avoid the thymic selection process that occurs during embryonic development. This is of great advantage for studying the immune responses against foreign antigens such as viral proteins since transgenic mice expressing viral genes are usually found to be tolerant to the viral gene products. We have developed two transgenic mouse models, each conditionally expressing SV-40 T antigen (Tag) or LacZ . To obtain these animals, we first generated transgenic mice expressing the tTA transactivator under the control of liver-specific albumin promoter (AlbtTA) or mouse urinary protein promoter (MuptTA) and then cross these animals with mice containing tetTag or tetLacZ transgene. Offsprings were genotyped and subjected to tetracycline (Tc) treatment by subcutaneous implantation of slow release Tc pellets. Expression of the transgene was analyzed 21 days after Tc implantation. Tag expression was detected in the liver of animals that were double-positive for either AlbtTA/tetTag or MuptTA/tetTag. In both cases transgene expression was confined to the liver only and could be completely abolished by Tc treatment as evidenced by absence of transgene transcription by RT-PCR. Tag protein synthesis resumed upon withdrawal of Tc. Similarly, expression of LacZ in mice that were double-positive for MuptTA/tetLacZ was tightly regulated by Tc treatment. 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. - Cytopathic Effect/Inducible Expression/Immune Response/Animal Model