【摘要】 利用CRISPR/Cas9系统构建敲除Prdx6基因的RAW264.7小鼠巨噬细胞系,为探讨Prdx6与布鲁菌感染及胞内寄生的关系构建细胞模型。针对Prdx6基因的第1外显子设计sgRNA,构建重组表达载体Prdx6-pX459-sgRNA,将重组质粒转染RAW264.7细胞,嘌呤霉素初步筛选。单细胞接种并扩繁,提取基因组DNA,PCR及测序鉴定。结果表明,在敲除细胞株基因组中存在碱基缺失,导致移码突变。说明成功获得敲除Prdx6基因的RAW264.7细胞系。更多还原

【Abstract】 This study aimed to establish a peroxiredoxin 6(Prdx6)knockout cell strain,and construct a cell model for exploring the effect of Prdx6 in Brucellainfection of RAW264.7 cells.The sgRNA was designed for the first exon of the Prdx6 gene,and the recombinant expression vector Prdx6-pX459-sgRNA was constructed.The recombinant plasmid of Prdx6-pX459-sgRNA was transfected into RAW264.7 cells,then the transfected cells were initially screened by puromycin.All the monoclones were cultured,and the genomic DNA was extracted from the cultured cells.Finally,the knockout results were identified by PCR amplification and sequencing.The sequencing results showed that a deletion-frameshift mutation was found in the genome of the knockout cell line.The results indicated that the Prdx6 knockout RAW264.7 cell line was established successfully.更多还原