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，我们能够诱导抗体和T细胞增殖反应，但不是细胞毒性T细胞反应，对HCV结构蛋白在这些转基因动物，这表明在T细胞水平的耐受性是更难打破。我们的实验室还产生表达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，并且还可以避免胚胎发育期间发生的胸腺选择过程。这对于研究针对外源抗原如病毒蛋白的免疫应答是非常有利的，因为通常发现表达病毒基因的转基因小鼠对病毒基因产物具有耐受性。我们已经开发了两种转基因小鼠模型，每一种都有条件地表达SV-40 T抗原（Tag）或LacZ。为了获得这些动物，我们首先产生在肝脏特异性白蛋白启动子（AlbtTA）或小鼠尿蛋白启动子（MuptTA）控制下表达tTA反式激活因子的转基因小鼠，然后将这些动物与含有tetTag或tetLacZ转基因的小鼠杂交。对后代进行基因分型，并通过皮下植入缓释Tc颗粒进行四环素（Tc）治疗。在Tc植入后21天分析转基因的表达。在AlbtTA/tetTag或MuptTA/tetTag双阳性动物的肝脏中检测到标签表达。在这两种情况下，转基因表达仅限于肝脏，并可以完全废除由Tc治疗证明了通过RT-PCR的转基因转录的情况下。标记蛋白质合成恢复后撤销Tc。类似地，LacZ在MuptTA/tetLacZ双阳性小鼠中的表达受到Tc治疗的严格调节。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。- 细胞病变效应/诱导表达/免疫应答/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