斑马鱼Slc26A4基因敲除品系的构建  

Establishment of Slc26A4 Knockout Zebrafish Lines

作　　者：赖若沙[1] 谢缤灵 邓慧玲 付贵芳 刘嘉[1] 伍伟景[1] 谢华平 谢鼎华[1] LAI Ruosha;XIE Bingling;DENG Huiling;FU Guifang;LIU Jia;WU Weijing;XIE Huaping;XIE Dinghua(Department of Otolaryngology-Head and Neck Surgery,Second Xiangya Hospital,Central South University,Changsha,Hunan 410011,China;Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation,Changsha,410081,China;Animal Nutrition and Human Health Laboratory,Changsha,410081,China;State Key Laboratory of Developmental Biology of Freshwater Fish,Changsha,410081,China)

机构地区：[1]中南大学湘雅二医院耳鼻咽喉科,长沙410011 [2]动物肠道功能调控湖南省重点实验室,长沙410081 [3]动物营养与人体健康实验室,长沙410081 [4]淡水鱼类发育生物学国家重点实验室,长沙410081

出　　处：《中华耳科学杂志》2021年第4期641-646,共6页Chinese Journal of Otology

基　　金：湖南省自然科学基金项目(2019JJ50860);湖南省卫生健康委科研计划项目(20200666);湖湘高层次人才聚集工程项目(2018RS3069);湖南省科技领军人才项目(2019RS3022);省计划创新引导计划(2018SK52513)。

摘　　要：目的SLC26A4是大前庭水管综合征(enlarged ves-tibular aqueduct syndrome,EVAS)的致病基因,也是中国第二位致聋基因。研究发现SLC26A4突变能够导致Pendred综合征(PS),包括甲状腺肿、听力丧失和前庭导水管增大,但其具体的分子机制尚不清楚。为了研究该基因在内耳发育中的具体分子作用机制,本文利用CRISPR/Cas9基因编辑技术构建斑马鱼Slc26A4基因敲除品系。方法首先通过分析软件筛选出Slc26A4基因的敲除位点,利用PCR技术扩增该基因的sgDNA,再以sgDNA为模板转录得到sgRNA,将sgRNA和Cas9蛋白共同注射到斑马鱼胚胎中。结果经基因型鉴定,CRISPR/Cas9系统对斑马鱼Slc26A4基因的敲除有效。但基因敲除不影响整体胚胎发育,斑马鱼外观未出现明显畸形。结论使用CRISPR/Cas9系统成功建立了斑马鱼Slc26A4基因敲除突变品系,为研究该基因在内耳发育过程中的作用奠定了基础。Objective SLC26A4 is the causative gene of enlarged vestibular aqueduct syndrome(EVAS)and the second most common deafness gene in China.It is expressed in many tissues in the early stage of human embryos,especially in the inner ear.It has been reported that SLC26A4 mutations can cause Pendred syndrome(PS),including goiter,hearing loss(Pendred,1896)and enlarged vestibular aqueduct,but the molecular mechanism is still unclear.In order to elucidate the molecular role of this gene in inner ear development,we used cloning free CRISPR/Cas9 gene editing technology to establish an Slc26A4 knockout zebrafish line.Methods Firstly,two knockout sites on the gene were determined by online screening analysis.Next,the template sgDNA of this gene was amplified by PCR(polymerase chain reaction).Then,the template sgDNA was transcribed into the sgRNA.Finally,the sgRNA and Cas9 protein were co-injected into 1-cell stage zebrafish embryos.Results The effectiveness of the CRISPR/Cas9 tool in knocking out Slc26A4 gene sites was confirmed.The gene knockout did not affect the whole embryo development and zebrafish did not show visible deformities.Conclusion An Slc26A4 knockout zebrafish line was established successfully,which has laid a foundation for studying the role of Slc26A4 in inner ear development.

关 键 词：斑马鱼 CRISPR/Cas9 SLC26A4基因 内耳发育 

分 类 号：R764[医药卫生—耳鼻咽喉科]