激光生物学报, 2018, 27 (3): 252, 网络出版: 2018-09-07
利用CRISPR/Cas9 技术建立斑马鱼Asb11基因敲除品系
Establishing the Asb11 Knockout Lines of Zebrafish with CRISPR/Cas9 Targeting Technology
摘要
Asb11基因被报道与斑马鱼Notch信号的激活有关,本研究室过去的研究显示该基因在心肌和骨骼肌中特异性表达。因此推测Asb11基因可能是心脏发育相关候选基因。为了阐明Asb11基因在斑马鱼心脏发育过程中的作用,本文利用CRISPR/Cas9打靶技术构建敲除Asb11基因的斑马鱼品系。首先在线分析筛选出Asb11基因最适合的打靶位点,然后PCR扩增出Asb11基因gRNA的双链cDNA,再将Asb11基因的gRNA和Hcas9的mRNA共同注射到斑马鱼胚胎Ⅰ细胞期胚胎中。进行打靶的有效性检测,发现Asb11基因的一号外显子出现了碱基的缺失,表明CRISPR/Cas9 系统对Asb11基因的敲除是有效的。对其F0代、F1代、F2代进行筛选,成功获得了Asb11基因敲除的斑马鱼品系,为探究Asb11在心脏发育中的作用奠定了基础。
Abstract
Asb11 gene was reported that it was associated with activation of Notch signaling pathway. The previous results in our lab showed that the gene expressed specifically in cardiac muscle and skeletal muscle. Therefore, it was hypothesized that Asb11 gene might be a candidate gene related to heart development. In this study, CRISPR/Cas9 targeting technology was employed to knockout zebrafish Asb11 gene. Firstly, the most suitable target sites in Asb11 gene were screened out on websites. Secondly, double-stranded DNA of the Asb11 gene targeting gRNA was amplified by PCR. Thirdly, the Asb11 gene gRNA and Hcas9 mRNA were co-injected into zebrafish embryos at 1-cell stage. The results of the effectiveness-test showed that bases were deleted in the exon1 of the zebrafish Asb11 gene, suggesting that the CRISPR/Cas9 system was effective for knockout of Asb11 gene. After screening in the F0, F1 and F2 generations, the lines of zebrafish with Asb11 gene knocked-out were established successfully. The work laid a foundation for exploring the roles of Asb11 in zebrafish heart development.
尹丽阳, 罗世锋, 陈宇, 漆轲婧, 彭云, 万永奇, 吴秀山, 李永青. 利用CRISPR/Cas9 技术建立斑马鱼Asb11基因敲除品系[J]. 激光生物学报, 2018, 27(3): 252. YIN Liyang, LUO Shifeng, CHEN Yu, QI Kejing, PENG Yun, WAN Yongqi, WU Xiushan, LI Yongqing. Establishing the Asb11 Knockout Lines of Zebrafish with CRISPR/Cas9 Targeting Technology[J]. Acta Laser Biology Sinica, 2018, 27(3): 252.