以RNF213基因创始者突变p.R4810K为TALENs核心靶点构建的新Moyamoya病斑马鱼遗传模型

项目名称： 以RNF213基因创始者突变p.R4810K为TALENs核心靶点构建的新Moyamoya病斑马鱼遗传模型

项目编号： No.81471180

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 盛文利

作者单位： 中山大学

项目金额： 70万元

中文摘要： 我们的前期实验发现，利用TALENs永久敲除Tg（flk-egfp）斑马鱼rnf213基因的1号外显子致移码突变后，导致颅内主干动脉血管壁不规则狭窄、颅内外异常芽生血管形成，与Moyamoya病（MMD）病理形态一致。进一步的Meta研究和生物信息学分析表明，p.R4810K突变严重影响RNF213基因的功能，是MMD创始者突变。在比较人类和斑马鱼的RNF213基因的同源保守序列后发现，两者的p.R4810K位点序列具有高度的相似性。在此基础上，利用TALENs技术，以p.R4810K为核心靶点，通过分别敲除rnf213基因第1、20、32外显子、敲入p.4810K及p.D4013N后构建相应的斑马鱼品系，蛋白芯片及泛素化分析探索变异位点体内致病机制，并通过双光子显微镜观察斑马鱼活体的血管表型，根据光镜和电镜病理、免疫组化、基因型改变优选出高度模拟人类MMD并能稳定传代的斑马鱼遗传模型。

中文关键词： 脑血管病；基因多态性；RNF；213基因；斑马鱼；遗传模型

英文摘要： Our previous studies have showed that knocked out permanently exon 1 of rnf213 in Tg(flk-egfp) transfected zebrafish with transcription activator-like effector nucleases (TALENs) caused irregular wall stenosis in intracranial trunk arteries and abnormal intracranial or extracranial sprouting vessels formation, which is consistent with Moyamoya disease(MMD) in pathology. Further meta-analysis and fundamental research confirmed that p.R4810K mutation as the founder mutation in MMD greatly influenced RNF213. Moreover, p.R4810K in zebrafish was remarkably similar to humans by comparing conservative homologous sequences of RNF213.On this basis, p.R4810K was confirmed as the core target site. Knocking out the the 1, 20, 32 exons of rnf213 individually and knocking in p.R4810K and p.D4013N by TALENs to explore the pathogenic mechanisms of these mutations in vivo though protein chip and ubiquitination analysis, and observe the vessel phenotypes with two-photon microscopy, calculate the pathological changes by immunohistochemical staining/optical microscope/electron microscope, and detect the genotypic changes in order to screen the best reproduceable and transmissible stably zebrafish genetic models highly imitating human MMD.

英文关键词： cerebral vascular diesese;gene polymorphism;RNF213 gene;zebrafish;genetic model