利用CRIS-PITCh系统构建原位表达CEP290-EGFP的细胞系  

Generation of CEP290-EGFP Knock-in Cell Line with the CRIS-PITCh System

作　　者：周士博 胡怀斌 涂海情 李慧艳 ZHOU Shi-bo;HU Huai-bin;TU Hai-qing;LI Hui-yan(National Center of Biomedical Analysis, Beijing 100850, China)

机构地区：[1]军事医学研究院生物医学分析中心,北京100850

出　　处：《科学技术与工程》2019年第11期35-39,共5页Science Technology and Engineering

基　　金：国家自然科学基金(81790252)资助

摘　　要：初级纤毛是一种存在于脊椎动物大多数细胞表面的细胞器,主要参与胚胎发育和传导胞外信号。在纤毛生长的过程中,过渡区关键蛋白CEP290主要定位在中心体上,而CEP290表达或定位异常影响初级纤毛的结构和功能。实验基于一种新型基因敲入技术——CRIS-PITCh系统,通过微同源介导末端连接方法,以实现将绿色荧光蛋白EGFP编码序列插入CEP290基因中,最终构建融合表达CEP290-EGFP蛋白的人视网膜色素上皮细胞系。因此,可以通过荧光成像直接观察内源CEP290的动态定位,为今后进一步研究CEP290的功能提供更好的工具和方法。Primary cilium is organelle present on most types of vertebrate cells, and it plays an important role during embryo development by transmitting extracellular signals. During the formation of primary cilia, CEP290 which has been reported to participate in the assembly of transition zone is mainly located in the centrosome. Additionally, depletion of CEP290 can destruct the structure and function of primary cilia. The study was based on a new knock-in system called CRIS- PITCh system which can insert EGFP in the end of CEP290 and make retinal pigment epithelial cell line express the CEP290-EGFP fusion protein. The fusion protein can be dynamically observed by EGFP fluorescence directly. It could provide a better method for further research on the function of CEP290.

关 键 词：初级纤毛 CRIS PITCh 系统 CEP290 视网膜色素上皮细胞 

分 类 号：Q291[生物学—细胞生物学]