斑马鱼adgrf3a基因敲除品系的构建

Construction of Zebrafish adgrf3a Gene Knockout Line

adgrf3a基因编码的ADGRF3A蛋白是黏附类G蛋白偶联受体(aGPCRs)家族的一员,主要在受精后5 d的胚胎以及成年斑马鱼的头部和性腺中表达。它含有一个GPS蛋白水解位点和7个跨膜结构域,与G蛋白相互作用行使其功能。斑马鱼是研究早期发育和成体内生理和病理的重要模式生物。为了研究adgrf3a在斑马鱼早期发育中的作用,我们利用CRISPR-Cas9技术构建了adgrf3a基因敲除斑马鱼品系。首先,通过分析软件筛选出该基因的敲除位点,利用聚合酶链式反应扩增该基因的向导DNA(sgDNA),再以sgDNA为模板进行体外转录得到向导RNA(sgRNA)并纯化回收,将纯化后的sgRNA和Cas9蛋白共同注射到斑马鱼1细胞期胚胎中,得到F0代嵌合体。随后,对斑马鱼胚胎进行基因编辑的有效性检测,结果表明,注射的胚胎出现了碱基缺失的现象,即sgRNA有效,将剩余胚胎培养至成鱼。将嵌合体与野生型斑马鱼杂交所得的F1代斑马鱼进行基因型鉴定,筛选adgrf3a突变杂合子,并对其adgrf3a突变位点进行Sanger测序,建立能够稳定遗传的adgrf3a基因杂合突变品系。之后,adgrf3a突变杂合子斑马鱼自交,获得adgrf3a纯合子突变斑马鱼。体视显微镜观察其成像发现,adgrf3a突变纯合子斑马鱼与野生型整体上未出现明显差异,然而,体内组织与器官的发育是否发生变化需要进一步验证。该基因敲除品系的建立为研究adgrf3a在早期发育及病理过程中的作用奠定了基础。

The ADGRF3A protein,encoded by the adgrf3a gene,is a member of the family of adhesion G protein-coupled receptors(aGPCRs)and is mainly expressed in embryos at 5 days after fertilization and in the head and gonads of adult zebrafish.It contains a GPS proteolytic site and seven transmembrane structural domains that interact with G proteins to perform its function.Zebrafish is a model organism for studying physiology and pathology during early development and adulthood.In order to study the role of adgrf3a in early zebrafish development,an adgrf3a knockout zebrafish line was constructed using CRISPR-Cas9 technology.Firstly,the knockout site of the gene was screened out by using analyzing software,and the guide DNA(sgDNA)was amplified by using polymerase chain reaction,next the guide RNA(sgRNA)was obtained by in vitro tran-scription and then the sgRNA and the Cas9 protein were co-injected into the zebrafish 1-cell-stage embryos.Then,the effectiveness of gene editing was tested,and the results showed that the injected embryos had base deletions,indicating that the gene knockout was successful.The F1 generation of zebrafish resulting from crosses between chimeras and wild-type zebrafish were genotyped,screened for adgrf3a mutant heterozygotes,and their adgrf3a mutant alleles were subjected to Sanger sequencing to determine the establishment of adgrf3a knockout lines.Subsequently,the adgrf3a mutant heterozygous zebrafish were selfcrossed to obtain adgrf3a mutant homozygous zebrafish.As observed and imaged by stereomicroscopy,the adgrf3a mutant zebrafish did not show a phenotype significantly different from that of the wild type.However,whether the development of tissues and organs in vivo is altered needs to be further verified.The establishment of this knockout line lays the foundation for exploring the role of adgrf3a in early development and pathology.