利用CRISPR/Cas9系统建立斑马鱼sonic hedgehog基因敲除模型及视网膜发育表型的初步鉴定

Establishment of sonic hedgehog gene knockout zebrafish model with CRISPR/Cas9 System and preliminary identification of the retinal developmental phenotype

目的:采用CRISPR/Cas9基因编辑技术建立sonic hedgehog(shh)基因敲除斑马鱼模型,并初步鉴定斑马鱼视网膜发育表型。方法:针对shh基因构建向导RNA(sgRNA)表达载体并转录,利用Cas9显微注射到斑马鱼受精卵中,经DNA鉴定及PCR扩增,初步鉴定基因编辑情况。观察基因敲除的斑马鱼形态,采用HE染色观察视网膜发育情况。结果:成功构建出shh杂交突变稳定遗传的斑马鱼种系,与野生型斑马鱼相比,shha+/-突变体胚胎及幼鱼多个系统的发育受到明显影响,出现鱼卵发育停滞、脊柱弯曲、心包水肿、眼球发育较小等多种发育异常。受精后96 h,HE染色发现视网膜神经节细胞及感光细胞排列紊乱,细胞数减少,胞核变大。结论:初步建立了shh基因敲除斑马鱼模型; shh基因敲除可引起斑马鱼视网膜发育异常,为进一步探讨斑马鱼shh基因对视网膜发育影响奠定了基础。

Objective:To establish a sonic hedgehog(shh)gene knockout zebrafish model using CRISPR/Cas9 technology,and preliminarily identify the retinal developmental phenotype.Methods:A gene knockout scheme was designed for shh gene,and a guide RNA(sgRNA)expression vector was constructed and transcribed.Cas9 was injected into zebrafish embryos.DNA identification and PCR amplification were conducted,and the gene editing was preliminarily identified.The morphology of gene knockout zebrafish was observed and the development of retina was observed by HE staining.Results:A stable inheritance of zebrafish shha hybridization mutation was successfully constructed.Compared with wildtype,the development of multiple systems of the shha+/-mutant embryo and juvenile was significantly affected,and there were various developmental abnormalities such as fish egg stasis,spine curvature,pericardium edema,and small eye development.HE staining showed that retinal ganglion cells and photoreceptor cells were disordered,the number of cells decreased and the nucleus became larger.Conclusion:A model of the Sonic Hedgehog(Shh)gene knockout zebrafish is preliminarily established.Shh gene knockout can cause abnormal retinal development of zebrafish,which lays a foundation for further study on the effect of shh gene on retinal development of zebrafish.