利用CRIS-PITCh系统构建原位表达CEP290-EGFP的细胞系

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。本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,我们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。

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

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. Our 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.