Cas9-CRISPR敲除hae3基因对斑马鱼血红蛋白生成的影响

Effect of knocking out hae3 gene using Cas9-CRISPR on hemoglobin of zebrafish Danio rerio

为获得hae3基因缺失的突变体,并研究hae3基因对血红蛋白生成的影响,以斑马鱼Danio rerio为研究对象,利用Cas9-CRISPR技术,成功建立了缺失血红蛋白hae3基因的鱼类模型,采用显微注射技术将hae3 sgRNA与Cas9 RNA混合后对斑马鱼第一细胞期受精卵动物极进行注射,利用序列测定与比对、T7核酸酶酶切方法成功检测到了hae3基因的敲除,并对缺失hae3基因的斑马鱼和对照组斑马鱼同时进行固蓝(O-dianisidine)染色。结果表明,与对照组相比,试验组斑马鱼血红蛋白含量明显下降,并有畸形表型。研究表明,胚胎时期hae3基因的缺失会严重影响斑马鱼血红蛋白的生成,而且伴随畸形发育表型。

In order to acquire hemoglobinless mutant and investigate the function of hae3 gene during hemoglobin generation,a model of hae3 gent was constructed using Cas9-CRISPR technique in zebrafish Danio rerio model which lacks of hae3 gene.Hae3 sgRNA and Cas9 RNA mixture were injected into the animal pole of zebrafish embryo with the first cleavage stage through microinjection,and identified by sequencing,multiple sequence alignment and T7 RNase digestion methods.The hemoglobin identification was conducted by O-dianisidine staining for both the hae3 knockout group and negative control.The results showed that the expression of hemoglobin was drastically decreased in the zebrafish which lacks hae3 gene and showed deformity phenotype compared with the wild type.Thus,we validate that lacking of hae3 gene severely affects the generation of hemoglobin and leads to developmental malformation during the embryonic period.