基于CRISPR/Cas9技术构建犬IRGM4、IRGM5和IRGM6基因敲除的MDCK细胞系

Knockout of IRGM4,IRGM5 or IRGM6 gene from MDCK cells using CRISPR/Cas9 technique

由干扰素诱导细胞产生的GTP蛋白酶M(IRGMs)在抵御病原微生物中发挥着核心作用。为建立IRGMs基因缺失的MDCK细胞系,本研究利用CRISPR/Cas9技术根据MDCK中IRGM4、IRGM5和IRGM6的基因序列设计并合成正链和负链引导RNA(gRNA)的靶DNA序列,以本实验室构建的含有单链引导RNA(sgRNA)骨架转录序列的通用质粒(p Gen-sgRNA)为模板,将U6启动子与合成的相关基因gRNA靶序列经PCR融合扩增,其产物再与sgRNA的骨架转录序列进行重叠延伸PCR扩增,并将扩增产物克隆于p GEM-T载体中,分别构建了具有靶向不同目的基因ORF的gRNA重组质粒。将这些重组质粒分别与pMJ920质粒共转染于MDCK细胞中,通过G418筛选和流式细胞仪分选,并以终点稀释法纯化培养筛选出的单细胞克隆株。经gRNA靶序列片段的测序鉴定,获得了目的基因敲除的MDCK细胞系,并分别命名为MDCK-IRGM4-、MDCK-IRGM5-和MDCKIRGM6-。通过western blot检测经γ干扰素诱导条件下的这3种IRGMs基因在敲除细胞系中相应的蛋白表达均为阴性。这3种MDCK细胞IRGMs敲除的细胞系的建立,为进一步研究IRGMs基因在抗细胞内病原微生物感染中的功能奠定了基础。

Immunity related GTPases M （IRGMs） induced by interferon play a pivotal role in resisting the infection of pathogenic microorganisms. To establish the 1RGMs knockout cell lines, three IRGMs （IRGM4, IRGM5 and IRGM6） knockout MDCK cell lines were prepared by CRISPR/Cas9 technique with the guide RNA （gRNA） designed according to positive and negative-strands of targeting genes of IRGM4, IRGM5 and IRGM6. In this procedure, the human U6 promoter controlled targeting gRNA sequence was fused with scaffold of guide RNA sequence by gene splicing by overlap extension PCR, respectively, which were cloned into pGEM-T vector to construct the gRNA plasmids. Then MDCK cells were co-transfected with each the gRNA plasmid and pMJ920, respectively. In addition, incubating in present of G418 for 48 hrs, the green fluorescence-positive cells in the transfected cells were sorted by flow cytometry and further identified by sequencing the knockout genes from the monoclonal cell lines, which were prepared by limited dilution. Those prepared IRGMs knockout cell lines were named MDCK-IRGM4-,MDCK-IRGM5- and MDCK-IRGM6-. Furthermore, no IRGM expression in 3 of the related the knockout cell lines induced by IFN--γ were detected by western blot, indicating the IRGMs knockout MDCK cell lines were successfully established, which would facilitate the further study about IRGMs genes in resisting pathogenic microorganism infection.