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Generation of a system enabling the study of hepatitis C entry factors in vivo and characterisation of HCV entry factor claudin-1

生成能够在体内研究丙型肝炎进入因子并表征 HCV 进入因子claudin-1的系统

基本信息

批准号：
238175417
负责人：
Dr. Markus von Schaewen
金额：
--
依托单位：
Department of Molecular Biology
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2014-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/238175417?language=en
关键词：
Generation system enabling study hepatitis

项目摘要

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. The standard therapeutic regime consists of pegylated interferon 2a/2b and ribavirin and depending on the genotype the protease inhibitors telaprevir or boceprevir.Despite all progress that has been done, treatment of HCV is rich of side effects and does not lead to a sustained virological response for a significant number of patients.An elemental prerequisite for the development of new therapeutic agents is the understanding of the viral life cycle. A crucial step is the virus entry into the hepatocyte.Until now seven (co-)receptors, that are involved in HCV cell entry (CD81, SCARB1, OLCN, NPC1L1, EGRF, EphA2 and CLDN1), have been identified.The proposed project aims to further elucidate the role of CLDN1 in HCV cell entry by using a genetically humanized mouse model. This model is based on the previous observation, that mice, which are naturally not susceptible for HCV, can be rendered permissive to cell culture derived HCV (HCVcc-FlpE) by adenoviral expression of human CD 81 and OCLN. The genome of HCVcc-FlpE encodes for a FlpE-recombinase and activates a yellow fluorescent protein reporter in Rosa26-FSF-F2AY reporter mice, which is used as a read-out for infection.This genetically humanized mouse model, which is well established in a modified form will now be combined with the technique of conditional gene ablation. The gerneration of mice with floxed exons in the CLDN1 locus is currently under progress. These mice will be crossed to an already existing deleter strain expressing CRE recombinase under an albumin promoter and leading to a liver-specific knockout of the CLDN1 gene. This step is necessary as a complete homozygous CLDN1 deficiency results in a neonatally lethal phenotype in mice.In the first instance we aim to characterize the hepatic CLDN1 deficiency. We will particularly address the question, how the expression profile of other members of the claudin family is altered and whether they can adopt the physiological role of CLDN1.The next step will be to cross the reporter mouse line with the liver-specific knockout of CLDN1. The resulting progeny will be transduced with human CD81 and OCLN and also with human or murine CLDN1. After injection of HCVcc-FlpE we will quantify reporter activity.This approach will allow to examine the impact of every given gene on HCV infection, even if the corresponding knockout would lead to a lethal phenotype.

丙型肝炎病毒（HCV）是世界范围内肝硬化和肝癌的主要原因。标准的治疗方案包括聚乙二醇干扰素2a/2b和利巴韦林，并根据基因型的蛋白酶抑制剂telaprevir或boceprevir。尽管已经取得了所有的进展，HCV的治疗是丰富的副作用，并没有导致持续的病毒学反应的显着数量的patients.An元素的先决条件，为新的治疗药物的发展是病毒的生命周期的理解。HCV进入肝细胞是病毒进入肝细胞的关键步骤，目前已鉴定出7种与HCV进入肝细胞有关的受体（CD 81，SCARB 1，OLCN，NPC 1 L1，EGRF，EphA 2和CLDN 1），本研究拟利用基因人源化小鼠模型进一步阐明CLDN 1在HCV进入肝细胞中的作用。该模型是基于先前的观察，即通过人CD 81和OCLN的腺病毒表达，可以使天然对HCV不敏感的小鼠对细胞培养物衍生的HCV（HCVcc-FlpE）具有容许性。HCVcc-FlpE基因组编码FlpE重组酶，并激活Rosa 26-FSF-F2 AY报告小鼠中的黄色荧光蛋白报告基因，其用作感染的读出。在CLDN 1基因座中具有floxed外显子的小鼠的生殖目前正在进行中。这些小鼠将与已经存在的在白蛋白启动子下表达CRE重组酶并导致CLDN 1基因的肝脏特异性敲除的删除菌株杂交。这一步是必要的，因为完全纯合的CLDN 1缺陷会导致小鼠的致死性表型。我们将特别解决这个问题，如何改变claudin家族的其他成员的表达谱，以及他们是否可以采用CLDN 1的生理作用。下一步将是交叉报道小鼠系与肝脏特异性敲除CLDN 1。所得子代将用人CD 81和OCLN以及用人或鼠CLDN 1转导。在注射HCVcc-FlpE后，我们将定量报告基因活性，这种方法将允许检查每个给定基因对HCV感染的影响，即使相应的敲除会导致致死表型。