Cas9技术在斑马鱼中鉴定MIP新突变位点功能

项目名称： Cas9技术在斑马鱼中鉴定MIP新突变位点功能

项目编号： No.81470619

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： 齐艳华

作者单位： 哈尔滨医科大学

项目金额： 73万元

中文摘要： Cas9/gRNA自2013年底开始成功应用于基因打靶，很快成为一种比核酸酶(ZFNs)和TALEN核酸酶更容易设计、特异性更高和毒性更低的打靶技术，是当前用于基因敲除动物制备的最先进的方法，特别是在斑马鱼中的应用。本课题申请人通过外显子组测序发现了基因MIP的一个新的突变可能导致一个三代中国人进展性先天性白内障的发生，这是世界上对该基因导致进展性先天性白内障的首次报道，但目前仍需通过基因敲除进一步验证该位点与先天性白内障的关系。晶状体膜蛋白MIP是含量最丰富的晶状体纤维细胞膜蛋白，对保持晶状体的透明性有十分重要的意义。本课题拟利用Cas9/gRNA技术，建立MIP基因敲除斑马鱼模型，从而利用光镜、电镜、免疫组化及分子生物学方式观察基因敲除后晶状体纤维的有序组织结构的变化和晶状体透明性的改变，并通过基因挽救证实其生物效应，从而研究该基因导致进展性先天性白内障的原因。

中文关键词： Cas9；斑马鱼；MIP；突变

英文摘要： As the new konckout technology, Cas9/gRNA（ guide-RNA）transcription activator-like effector nucleases (TALENs) is simpler and less toxicity than zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).Cas9/gRNA has been valued and applied widely in the field of genetics, including the genetic eye disorders. A collection of species and genes that have been successfully modified by Cas9/gRNA, especially in zebrafish. A novol MIP mutation was found in a three generation Chinese progressive congenital cataract family in our recent exome sequencing research. This is the first mutation in the MIP gene has been reported cause progressive congenital cataract in the world. However, this result still needs knockout technology to confirm further. Therefore, we will perform further study of the new valiation in zebrafish using TALE nuclease engineering including targeted genome modification、homologous recombination、gene rescue experiment and whole mount in situ hybridization.We will still perform light microscopy, scanning electron microscopy, freeze-fracture transmission electron microscopy immunohistochemistry and molecular biological technology on the MIP knockout zebrafish to understand the protein well in the lens. In recent years, the studies reveals MIP plays a crucial role in the development of the lens. However, the exactly mechanism of MIP mutation in progressive congenital cataract is still unclear. This mechanism may be more useful in age related cataract research. Therefore, this study is important and necessary.

英文关键词： Cas9;zebrafish;MIP;mutation