利用CRISPR/Cas9技术制备Mindin基因敲除小鼠  

Application of CRISPR/Cas9 Technology for Establishing Mindin Gene Knockout Mice

作　　者：纪慈数[1] 赵志刚[2] 贺颖[1] 

机构地区：[1]厦门大学实验动物中心,福建厦门361102 [2]广州军区武汉总医院,湖北武汉430000

出　　处：《厦门大学学报（自然科学版）》2016年第6期922-926,共5页Journal of Xiamen University：Natural Science

基　　金：福建省自然科学基金重点项目(2013Y01010490)

摘　　要：针对Mindin基因编码序列设计3条单链向导RNA(sgRNA),制备相应质粒,将表达sgRNA和Cas9蛋白的质粒共同电转入小鼠成纤维细胞L929中,通过药物筛选获得细胞库.经单克隆测序检测细胞库中基因组的突变效率,选择效率最佳的sgRNA.体外转录获取sgRNA和Cas9mRNA,进行小鼠受精卵的显微注射后提取小鼠基因组DNA测序鉴定.产生的40只小鼠中17只发生不同程度的碱基插入或缺失,其中23号小鼠缺失10个碱基造成移码突变,Mindin基因表达被破坏,成功构建出Mindin基因敲除小鼠,为深入研究Mindin基因的生物学功能奠定了基础.To establish Mindin knockout mice using CRISPR/Cas9 gene targeting technology,three sgRNA targets of Mindin were designed according to Mindin genome sequence.Vectors expressing sgRNA and Cas9 were constructed separately,and co-transfected into L929 cells by with electroporation method.Transfected cells were selected with blasticitin and the mutation rate was determined using DNA sequencing with proper primers. The sgRNA which had resulted in the highest mutation rate in L929 cells was transcribed in vitro ,along with Cas9.Then sgRNA and Cas9 mRNAs were microinjected into mice zygotes and 40 mice were born after the microinjection.Genotypes of the mice were determined by PCR with genomic DNA from the new born mice tail and 17 neonatal mice were found carrying mutations in Mindin gene by subsequent sequencing.Gene sequencing results showed that all mutant mice had different levels of nucleotide insertion or deletion,with the maximum number of 10 base deletion found in mouse No.23,leading to the reading frame shift and Mindin gene silence.Taking together,we have successfully established a mouse model with Mindin gene knockout,which lays a solid foundation for related future research.

关 键 词：CRISPR/Cas9 基因敲除 Mindin 

分 类 号：Q789[生物学—分子生物学]