ucp2基因敲除斑马鱼的模型建立及其铝暴露神经毒性相关研究  

Effects of knockout of ucp2 gene on zebrafish dementia model induced by aluminum

作　　者：汪雪雁[1] 谈勇 张芳 陈豪 贺亚南 李行舟 赵宝全 WANG Xueyan;TAN Yong;ZHANG Fang;CHEN Hao;HE Yanan;LI Xingzhou;ZHAO Baoquan(School of Tea and Food Science&Technology,Anhui Agricultural University,Hefei 230036;China Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850)

机构地区：[1]安徽农业大学茶与食品科技学院,合肥230036 [2]军事科学院军事医学研究院毒物药物研究所,北京100850

出　　处：《安徽农业大学学报》2021年第3期397-402,共6页Journal of Anhui Agricultural University

基　　金：国家“重大新药创制”科技重大专项(2018ZX09J18102-001-003)资助。

摘　　要：利用CRISPR/Cas9技术敲除斑马鱼ucp2基因,研究ucp2基因在铝致斑马鱼痴呆中的作用。针对斑马鱼ucp2基因设计并制备gRNA,通过显微注射技术将gRNA与Cas9-mRNA混合注入斑马鱼单细胞胚胎中,利用酶切和基因测序筛选出发生突变的F0代斑马鱼,与野生型斑马鱼杂交得到F1代斑马鱼杂合子,分析其突变类型,将突变类型一致的斑马鱼杂合子杂交,获得F2代。在酸性AlCl3中暴露F2代斑马鱼胚胎,暴露组的斑马鱼个体行为出现异常,移动距离、移动速度和移动频率皆显著下降(P<0.01),体内活性氧(ROS)水平显著升高(P<0.01);其中KO+AlCl3组导致的异常更为明显,psen1、psen2、tnf-α和il-1βmRNA转录水平显著上调(P<0.01)。ucp2在铝致斑马鱼痴呆中起到重要作用,可能是其关键的治疗预防靶点。In order to explore the role of ucp2 gene in aluminum-induced zebrafish dementia,we established ucp2 gene knockout zebrafish model on wild type zebrafish using CRISPR/cas9 system.We selected the ucp2-targeting region by bioinformatics analysis,constructed and transcribed gRNA plasmid in vitro,and con-injected mixed gRNA and Cas9 mRNA into single-cell stage fertilized embryo.We screened out the mutant F0 generation zebrafish by enzyme digestion and Sanger sequencing,chose mutant successfully F0 outcrossed with the wild-type zebrafish to get the F1 generation zebrafish,and used the same methods to get F2 generation zebrafish.The larvae of F2 zebrafish were exposed in acidic AlCl3.Compared with the control group,the individual behavior of the zebrafish exposed to AlCl3 were abnormal,the distance,speed and frequency of swim for zebrafish significantly decreased(P<0.01),while the ROS level was significantly increased(P<0.01),and the mRNA transcription levels of psen1,psen2,tnf-αand IL-1βwere up-regulated significantly(P<0.01).In conclusion,we successfully established ucp2 gene knockout zebrafish model,which could speed up the process of AD(Alzheimer’s disease).

关 键 词：CRISPR/Cas9 斑马鱼 ucp2基因 阿尔茨海默病 铝 

分 类 号：S917[农业科学—水产科学]