利用CRISPR/Cas9构建稳定遗传的斑马鱼TYRO3纯合突变体

Construction of zebrafish TYRO3 homozygous mutants using CRISPR/Cas9 system

目的:本研究利用CRISPR/Cas9构建稳定遗传的斑马鱼TYRO3纯合突变体,为利用斑马鱼研究TYRO3功能构建模型。方法:通过PCR和测序鉴定基因型,筛选获得TYRO3基因突变斑马鱼。通过观察斑马鱼水肿的表型以及用透射电镜观察足突融合的情况了解TYRO3突变的影响。结果:我们得到了两个移码的TYRO3突变体,每个TYRO3突变体在F2中获得2和5个纯合子,并且F0杂合子突变率可达50%。TYRO3基因突变斑马鱼出现卵黄囊、心包及眼周水肿,电镜下出现明显的足突融合。结论:本研究成功构建了TYRO3基因突变的纯合子斑马鱼,并初步证实,TYRO3突变会导致斑马鱼肾脏足细胞损伤。

Objective:In this study,we chose zebrafish as our model organism and constructed TYRO3 homozygous mutant which was helpful to explore change of its function caused by TYRO3 knock-out using CRISPR/Cas9 system.Methodology:PCR and sequencing were used to identify the mutations in the embryo.The effects of TYRO3 mutants on zebrafish edema were observed,as well as foot process effacement visualizing by transmission electron microscopy.Results:We got two frameshift TYRO3 mutants,2 and 5 homozygous for each TYRO3 mutants were acquired in F2.Heterozygous rate was 50%in F0.TYRO3 mutants lead to impaired glomerular filtration barrier(GFB),as presented by pericardial and periorbital edema,and podocyte foot-process effacement.Conclusion:We successfully constructed a homozygous zebrafish with gene TYRO3 knockout and preliminarily confirmed that TYRO3 deletion could cause damage to zebrafish podocytes injury.