利用CRISPR/Cas9技术构建斑马鱼Hand2基因敲除模型

Construction of the Zebrafish Hand2 Gene-knockout Line by Using CRISPR/Cas9

心脏神经脊衍生物表达转录因子2(Hand2)是bHLH家族的成员,也称为dHand、Hed和Thing2,可在人以及鼠、鸡、斑马鱼、果蝇等模式动物中表达。国内外的研究显示,Hand2主要影响右心室的发育,在维持右心室祖细胞和右心室形态发生中起关键作用。为了进一步研究Hand2基因参与心脏发育的作用机制,拟通过CRISPR/Cas9基因编辑技术构建斑马鱼Hand2基因敲除品系。根据生物信息学网站的分析筛选出最优的两个靶位点,设计并合成引物,利用PCR扩增得到Hand2基因sgRNA的cDNA,再转录为mRNA,将两个靶位点sgRNA的mRNA和Cas9蛋白共同注射到斑马鱼胚胎的Ⅰ-细胞期。在斑马鱼的胚胎和成鱼时期进行基因敲除的有效性检测,发现在靶位点附近有碱基的缺失和插入,表明CRISPR/Cas9对斑马鱼Hand2基因敲除是有效的。本研究利用CRISPR/Cas9基因编辑技术构建了斑马鱼的Hand2基因敲除模型,为后期研究Hand2基因在人类疾病中的作用机制和新型治疗靶点的临床开发等奠定了基础。

Heart and neural crest derivatives expressed transcription factor 2(Hand2)is a member of bHLH family,also known as dHand,Hed and Thing2,which can be expressed in human,mouse,chicken,zebrafish,Drosophila and other model animals.Domestic and foreign studies have shown that Hand2 mainly affects the development of the right ventricle and plays a key role in the maintenance of right ventricular progenitor cells and morphogenesis of the right ventricle.In order to further study the mechanism of Hand2 gene involved in heart development,a zebrafish Hand2 gene knockout lines was constructed by CRISPR/Cas9 gene editing technology.According to the analysis of bioinformation website,the optimal two target sites were screened,and primers were designed and synthesized.The cDNA of the Hand2 gene sgRNA was obtained by PCR amplification,and then transcribed into mRNA.The mRNA of the two target sites sgRNA and Cas9 protein were co-injected into theⅠ-cell stage of zebrafish embryos.The effectiveness of gene knockout was tested in zebrafish embryos and adults,and it was found that there were base deletions and insertions near the target site,indicating that CRISPR/Cas9 was effective for zebrafish Hand2 knockout.In this study,a knockout model of Hand2 gene in zebrafish was constructed using CRISPR/Cas9 gene editing technology,which laid a foundation for the later study of the mechanism of Hand2 gene in human diseases and the clinical development of novel therapeutic targets.