糖原贮积症0型斑马鱼疾病模型的构建及表型分析  

Establishment and phenotypic analysis of a zebrafish disease model of glycogen storage disease type 0

作　　者：黄姣 霍锦倩 刘姣 陈洁[1,2,3] 祖尧[1,2,3] 张庆华 任建峰[1,2,3] HUANG Jiao;HUO Jinqian;LIU Jiao;CHEN Jie;ZU Yao;ZHANG Qinghua;REN Jianfeng(International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306)

机构地区：[1]上海海洋大学科技部海洋生物科学国际联合研究中心,上海201306 [2]上海海洋大学水产种质资源发掘与利用教育部重点实验室,上海201306 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《中国比较医学杂志》2021年第12期43-52,共10页Chinese Journal of Comparative Medicine

基　　金：上海市科委国际科技合作项目(15410723300)。

摘　　要：目的利用CRISPR/Cas9基因编辑技术构建糖原贮积症0(GSD0)型斑马鱼疾病模型,研究该病的发病进程和机制。方法针对gys1和gys2基因各设计一个靶点,进行基因编辑和突变体筛选;利用qPCR技术检测早期发育阶段和成鱼不同组织基因表达情况,通过PAS染色技术检测糖原累积情况。结果获得了gys1和gys2基因各两种有效突变类型的F2纯合突变体,成功构建了GSD0型斑马鱼疾病模型。gys^(1-/-)纯合子F3早期胚胎发育迟缓,48 hpf内全部死亡,而gys^(2-/-)纯合子发育正常。基因表达分析显示,野生型斑马鱼早期发育阶段(0~125 hpf),gys1和gys2的表达先下降后上升,受精卵时期表达最高;成鱼阶段,gys1在心脏和肌肉组织中高表达,gys2在肝中高表达。PAS糖原染色显示,与野生型相比,gys^(1-/-)心脏和肌肉没有明显的糖原积累,gys^(^(2-/-))肝没有明显的糖原积累。结论gys1和gys2基因敲除斑马鱼模型的表型与小鼠模型相似,但也存在差异;Gys1敲除小鼠模型中F2大部分纯合子死亡,斑马鱼gys^(^(1-/-))纯合子F2正常生长发育,而自交产生的F3不能存活。斑马鱼gys1和gys2敲除突变体的制备,为进一步研究人类糖原储存障碍GSD0的发病进程和机制提供了材料。Objective To study the detailed process and mechanism of glycogen storage disease(GSD)type 0 using a zebrafish disease model established by CRISPR/Cas9 gene editing technology.Methods Target sites were determined for gys1 and gys2.After gene editing,homozygous mutants were screened.The expression levels of gys1 and gys2 were quantified with qPCR at early developmental and adult stages.Glycogen accumulation was assessed by periodic acid-Schiff staining.Results Homozygous F2 gys1 and gys2 genes with two effective mutation types were successfully obtained.Homozygous gys^(1-/-)F3 had a delay in early embryonic development and died within 48 hpf,whereas homozygousgys^(2-/-)developed normally.Gene expression analyses of gys1 and gys2 in wildtype zebrafish showed that their expression levels decreased first and then increased during early developmental stages(0~125 hpf)with the highest expression at the fertilized egg stage.At the adult stage,gys1 was highly expressed in heart and muscle tissues,while gys2 was highly expressed in the liver.Glycogen staining showed no significant glycogen accumulation in the heart or muscle of gys^(1-/-)or in the liver of gys^(2-/-)compared with the corresponding wildtype tissues.Conclusions The phenotypes of gys1 and gys2 knockout zebrafish models were similar to those of mouse models,but there were some differences.In the Gys1 knockout mouse model,most F2 homozygotes die.Among gys1 knockout zebrafish,gys^(1-/-)homozygous F2 developed normally,whereas F3 generated by self-crossing did not survive.The model of gys1 and gys2 knockout in zebrafish provides materials for further study on the pathogenesis of human GSD type 0.

关 键 词：糖原贮积症GSD0 疾病模型 斑马鱼 基因敲除 gys1 gys2 

分 类 号：R-33[医药卫生]