108M780-上海海洋大学学报.DOC

利用CRISPR/Cas9 敲除斑马鱼细胞周期蛋白M2（CNNM2）基因 黄振玉，竹个个，邹华锋，吕为群 (上海海洋大学 水产与生命学院，上海 201306) 摘要：细胞周期蛋白M2（CNNM2）是参与体内镁离子平衡的重要候选基因，实验采用的CRISPR/Cas9 系统是近几年发展起来的一类新型基因打靶技术。在斑马鱼CNNM2基因的一号外显子处选取打靶位点，构建sgRNA表达载体。随后将体外转录的sgRNA和Cas9 mRNA混合物分别以约30pg和300pg的剂量显微注射到斑马鱼单细胞期的受精卵中实现对目的靶基因CNNM2的敲除。24~48h后PCR产物回收，限制性酶切法初步检测基因打靶情况，最后进行测序分析确定突变类型。为进一步验证突变的稳定遗传性，选择了具有敲除效率F0与野生型斑马鱼进行外交，对获得的F1进行逐一剪尾，提取DNA进行PCR和测序分析。结果显示F0和F1均获得了不同程度插入，缺失和移码突变类型，且F0突变效率为88%。本研究获得了能稳定遗传的CNNM2基因敲除斑马鱼个体，并为将来进一步研究CNNM2基因和机体镁离子稳态之间的关系打下了基础。 关键词：CRISPR/Cas9；CNNM2；斑马鱼；基因敲除 研究亮点：目前关于离子调控的研究主要集中在钠、钾和钙离子上，有关镁离子在鱼类发育过程中的生理功能和作用机制的研究还相对缺乏。本实验利用CRISPR/Cas9系统敲除斑马鱼中参与镁离子调控的重要候选基因CNNM2，获得能稳定遗传的敲除个体。为深入研究CNNM2基因在镁离子调控中的生理功能及调控机制提供基础材料。 Generating Cyclin M2 (CNNM2) knockout zebrafish using CRISPR/Cas9 system HUANG Zhenyu, ZHU Gege, ZOU Huafeng,LüWeiqun (College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China) Abstract：CyclinM2(CNNM2) is one of the regulatory transport genes that involved in zebrafish Mg2+ homeostasis. We generated CNNM2 gene knockout zebrafish by CRISPR/Cas9 system which had been developed into a novel gene targeting system in recent years．First we constructed the sgRNA expression vector targeting zebrafish CNNM2 gene. Then after in vitro transcription of sgRNA and Cas9 vectors, we co-injected them into one-cell fertilized eggs of zebrafish to generate zebrafish with targeted mutation. After 24h-48h, injected embryos were collected and used to conduct mutation analysis with restriction endonuclease and sequencing. To further verify the stability of genetic mutations, we selected mutated founder to outcross with wild type zebrafish. a knockout and wild-type diplomatic F0 efficiency of were the obtained, and then F1 were analyzed by PCR and sequencing of the caudal fin. The results showed F0 and F1 were introduced insertion, deletion and frameshift mutations to some extend, and F0 mutation efficiency was 88%. The CNNM2 gene knockout zebrafish generated in this study can be transmitted by g