CRISPRCas9系统对斑马鱼fhl1a基因敲除有效性研究.docVIP

CRISPRCas9系统对斑马鱼fhl1a基因敲除有效性研究 　　摘 要 为研究fhl1a基因在心脏发育中的作用，利用CRISPR/Cas9系统敲除斑马鱼fhl1a基因.在斑马鱼fhl1a基因的三号外显子处选取打靶位点，构建sgRNA表达载体.将体外转录的sgRNA和Cas9 mRNA混合物分别显微注射到斑马鱼单细胞期的受精卵中以期实现对目的靶基因fhl1a的敲除.随后收集部分72 h胚胎进行PCR检测和产物直接测序，确定有无突变.为进一步验证突变是否可稳定遗传，选择具有敲除了的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F1获得了不同程度的插入、缺失等突变类型.研究结果证明所设计的fhl1a基因CRISPR/Cas9敲除系统能在斑马鱼体内有效地形成基因突变，该突变能稳定地遗传到下一代个体，这为进一步研究fhl1a基因在心脏发育中的具体调控机制打下了基础. 　　关键词 CRISPR/Cas9；fhl1a基因；斑马鱼；敲除 　　中图分类号 Q812 文献标识码 A 文章编号 1000-2537（2017）03-0021-06 　　The Validation Study of Zebrafish Fhl1a Gene Knockout by CRISPR/Cas9 System 　　CHEN Si-xing， CHEN Fa， CAI Wan-wan， WU Yan， LIAO Yan-wei， DENG Yun， WU Xiu-shan， WANG Yue-qun* 　　（The Center for Heart Development， Key lab of MOE for Department Biology and Protein Chemistry， Hunan Normal University， Changsha 410081， China） 　　Abstract To study the function of the fhl1a gene in heart development， we generated the fhl1a gene knockout zebrafish using the CRISPR/Cas9 system. At first， we constructed the sgRNA expression vector targeting zebrafish fhl1a gene at the third exon. After in vitro transcription of sgRNA and Cas9 vectors， we co-injected them into single-cell fertilized zebrafish eggs to generate zebrafish with the targeted mutation. After 72 h of injection， embryos were collected， the genome DNA was extracted， PCR and sequencing were then performed. To further verify the stability of genetic mutations， we selected mutated founder to outcross with wild type zebrafish. The first generation （F1） was then analyzed by PCR and sequencing of the caudal fin. Our results confirmed that insertion and deletion mutations were able to be introduced into F1 using this method. Our study demonstrated that CRISPR/Cas9-induced fhl1a gene mutations can be successfully transmitted through the gremline， which lays the foundation for future studies on gene fhl1as specific regulatory mechanism during heart development. 　　Key words CRISPR/Cas9；fhl1a gene；zebrafish；knockout 　　斑?R鱼因其成年鱼个体小、易于饲养、发