基质金属蛋白酶-9基因敲除斑马鱼模型的建立

Establishment of zebrafish (Danio rerio) model with the knockout of matrix metalloproteinase-9 gene

目的建立斑马鱼基质金属蛋白酶-9(MMP9)基因敲除模型,为研究MMP9基因在阿尔茨海默病(AD)中的作用机制奠定基础。方法根据CRISPR/Cas9的靶点设计原则,利用生物信息学方法,选取MMP9基因敲除靶点,构建gRNA质粒,将gRNA和Cas9的mRNA混合后注入AB野生型和APPsw转基因斑马鱼(APPsw+/+)的受精卵单细胞中,48 h后通过PCR、酶切及基因测序等方法验证基因敲除情况;遗传学方法传代筛选,建立斑马鱼mmp9-/-、APPsw+/+-mmp9-/-基因敲除品系;对基因敲除品系的发育、突变遗传率、突变类型和行为学等进行研究。结果构建成功4条gRNA,m2是最佳敲除靶点,突变遗传率平均>50%;野生型和APPsw转基因斑马鱼模型分别获得2个突变类型,APPsw+/+-mmp9-/-模型分别在敲除部位缺失4个和8个碱基,mmp9-/-缺失5个碱基和敲入5个碱基并伴有1处点突变,建立了野生型和APPsw过表达型MMP9基因敲除斑马鱼品系;F2代纯合子孵化率、幼鱼期表型和行为学检测结果与正常斑马鱼无统计学差异(P>0.05),成鱼期开始出现学习记忆能力的变化。结论MMP9基因的敲除能够减缓痴呆的发生发展进程,该模型的建立为AD研究提供支持。

Objective To establish matrix metalloproteinase-9(MMP9)gene knockout zebrafish(Danio rerio)model on the basis of wild type and APPswoverexpression zebrafish using CRISPR/Cas9 system.Methods The mmp9-targeting region was selected by bioinformatics analysis;gRNA plasmid was constructed and transcribed in vitro.The gRNA and Cas9 mRNA were mixed and then injected into one-cell stage fertilized embryo.Genomic DNA was extracted after 48 h,the target fragment was amplified by PCR,and than the effect was evaluated by T7 EI enzyme digestion and sanger sequencing.The classical genetic approach was used to screen mmp9-/-,APPsw+/+-mmp9-/-zebrafish strains.The development of the knockout strain,as well as the mutation heritability and mutation type were analyzed.Results Four gRNAs were successfully constructed,and m2 and Cas9 mRNA were chosen to inject.T7 EI digestion and DNA sequencing showed that the MMP9 was successfully edited by m2.The mutational heritability of the two models was over 50%and two mutation types were obtained for the two models,respectively.In zebrafish juveniles the hatching rate as well as the phenotype in the two knockout models were not significantly different from those of the control(P>0.05),while the behavior was changed in adult.Conclusion The MMP9 knockout can slow the progression of Alzheimer′s disease(AD),and the creation of the present model provides an animal model support for future AD studies.