CRISPR/Cas9系统介导的miR-155基因敲除细胞的制备

Preparation of miR-155 Knockout Cells Mediated by CRISPR/Cas9 Technology

为研究miR-155基因在猪肺泡巨噬细胞系(3D4/21)中的功能,采用CRISPR/Cas9系统构建miR-155敲除的猪肺泡巨噬细胞系。首先,采用sgRNA-cas9程序,设计4个靶向miR-155的特异性sgRNA引物序列。然后构建sgRNA表达载体,与pEGFP-C1-cas9载体共转染3D4/21细胞,进行流式分选并富集表达绿色荧光蛋白GFP的细胞。最后,采用T7E I酶酶切、Sanger测序、实时荧光定量PCR(qPCR)、western blot等方法检测敲除效率,发现miR-155基因可以被以上4个sgRNA编辑,其中sgRNA39的敲除效率最高,T7E I酶酶切检测流式分选后的敲除效率有42%;Sanger测序显示sgRNA39细胞系敲除31个碱基;RT-qPCR结果显示miR-155-5p/3p表达量均有下降;western blot结果表明miR-155靶基因SHIP1的蛋白表达量升高。成功构建miR-155敲除的3D4/21细胞为进一步探讨miR-155在巨噬细胞发挥的调控功能研究提供细胞模型。

To study the function of miR-155 in porcine alveolar macrophage cell line(3 D4/21),miR-155 knockout cells were constructed using CRISPR/Cas9 technology.Firstly,4 prime series specific to sgRNAs for targeting miR-155 were designed using sgRNAcas9 program.Then,sgRNA expression vectors were constructed and co-transfected with pEGFP-C1-cas9 vector into 3 D4/21 cells.Next,the cells expressing green fluorescent protein(GFP)were enriched and selected using the FACS-sorting strategy.Finally,T7 E I cleavage assay,Sanger sequencing,real-time quantitative PCR(RT-qPCR)and Western blot were used to detect the efficiency of the knockout.We found that miR-155’s genomic sequence was edited using 4 different sgRNAs,while the highest efficiency of knockout reached by sgRNA39,up to 42%after T7 EI enzyme digestion and FACS-sorting.Sanger sequencing demonstrated that 31 nucleotides of sgRNA39 cell line were knocked out.The RT-qPCR result showed that the expression levels of miR-155-5 p/3 p significantly decreased.The Western blot result indicated that the protein expression level of target gene SHIP1 in miR-155 significantly increased.To sum up,the successful establishment of the miR-155 knockout cell line provides cell model for further studying the regulation function of miR-155 in macrophage cells.