肝脏特异性可调控丙型肝炎病毒核心蛋白表达的转基因小鼠模型的建立

Development of Transgenic Mice Specially Express HCV-C in Liver by Tet-on System

【目的】制备TRE-HCV-C转基因小鼠,为四环素调控系统的体内研究提供了反应部分的转基因小鼠,以便与本实验室同时建立的调控部分的小鼠共同作用,为进一步建立双转基因小鼠模型奠定基础,更为丙型肝炎病毒核心蛋白(HCV-C)的发病机制的研究提供一个实用方便的模型。【方法】重组构建含有目的基因HCV-C、TRE序列和SV40 polyA的转基因载体pTRE-HCV-C,以显微注射的方法将1.153kb的转基因片段注入BALB/C母鼠的受精卵,出生动物及其后代经PCR初步筛选出阳性,再经Southern杂交对阳性鼠基因组DNA标本行进一步鉴定。用转基因阳性小鼠和整合有肝脏特异性启动子ApoE和rtTA基因的另一品系转基因小鼠杂交,得到子代F1小鼠,通过免疫组织化学来初步检测HCV-C在肝脏中特异性的可调控性的表达。【结果】产生了5只整合有TRE-HCV-C基因的首建鼠,以及它的子代也带有此基因。与基因组上整合有肝脏特异性启动子ApoE和rtTA基因的转基因小鼠杂交后,得到子代F1小鼠,特定时间与DOX作用后,小鼠肝脏的免疫组织化学结果表明,TRE-HCV-C小鼠为丙型肝炎病毒核心蛋白的发病机制研究提供了一个良好的动物模型工具。【结论】成功制备了HCV-C转基因小鼠,可利用四环素调控系统来研究HCV中的C基因对小鼠的作用,为进一步建立四环素调控系统调控下表达HCV-C基因双转基因小鼠模型奠定基础,也是HCV-C基因功能研究及与肝细胞癌的关系的机制研究的一个有用工具。

[Objective] To study the role of HCV core protein（HCV-C） in the pathogenesis of HCV disease by establishing TRE-HCV-C transgenic mice model mediated by Tet-On system, which will provide a powerful animal model for exploring the role of HCV-C in the etiology pathomorphology and treatment of HCV diseases. [ Methods ] For overcoming the leaky of adequate HCV animal model, a transgenic construct, containing HCV-C,TRE,the CMV min promter and the SV40 poly A, was generated, in which the HCV-C was isolated from the HCV cDNA library with PCR-screening. 1.513kb of DNA fragment was introduced into fertilized eggs by microinjection. Injected eggs were implanted into the oviducts of female mice respectively, from which offspring were obtained. The founder mice and their progeny were screened for integration of transgene into the mouse genome. Mating the two positive types of mice, identification and expression of ApoE-rtTA/TRE-HCV-C gene in their progeny were determined by PCR, Southern blot, and then after adding doxycycline （Dox） to the positive transgenic mice of two months old, HE staining, immuno-histochemistry and Western blot were performed. [Results] Five mice canT the HCV-C gene by the identification of PCR and Southern blotting. The transgenic F1 mice were crossed with mice whose genomic DNA is integrated by rtTA and ApoE canting construct. The results of ICC showed that the HCV-C protein was expressed in dual transgenic mice under the reaction of rtTA protein. [Conclusions] A transgenic mice model inducible expressed HCV core protein is established successfully, which can be used to study the role of HCV Core protein （HCV-C） in the pathogenesis of HCV disease, and to further lay the foundation of establishing dual transgenic mice model.