基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系  

Construction of Chicken Embryo Fibroblast Cell Line with TET2 Gene Knockout Based on CRISPR-Cas9 Technology

作　　者：王强州 潘诗雨 方梦雅 李微 王佳兴 刘茵茵[3] 陈世豪 WANG Qiangzhou;PAN Shiyu;FANG Mengya;LI Wei;WANG Jiaxing;LIU Yinyin;CHEN Shihao(Institute of Epigenetics and Epigenomics,Yangzhou University,Jiangsu Yangzhou 225009,China;College of Veterinary Medicine,Yangzhou University,Jiangsu Yangzhou 225009,China;Jiangsu Institute of Poultry Sciences,Jiangsu Yangzhou 225125,China)

机构地区：[1]扬州大学表观遗传学与表观基因组学研究所,江苏扬州225009 [2]扬州大学兽医学院,江苏扬州225009 [3]江苏省家禽科学研究所,江苏扬州225125

出　　处：《中国农业科技导报》2023年第11期227-233,共7页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(32002271);江苏省高等学校大学生创新创业训练计划项目(202211117101Y)。

摘　　要：为研究甲基胞嘧啶双加氧酶2(tet methylcytosine dioxygenase 2,TET2)及其介导的羟甲基化修饰在调控天然免疫反应中的作用,利用CRISPR-Cas9基因编辑技术构建TET2基因敲除的鸡胚成纤维细胞系。针对鸡TET2 mRNA的2种剪接变体的共有CDS序列设计4组sgRNA,构建sgRNA表达质粒,连同Cas9-T2A-GFP质粒共转染DF-1细胞,筛选高切割效率的sgRNA;将转染该sgRNA的DF-1细胞,进行流式细胞分选,获取表达绿色荧光蛋白(green fluorescent protein,GFP)的细胞,再通过有限稀释法筛选单克隆细胞,并用DNA测序进行鉴定。单克隆敲除细胞扩大培养后,Western blot和Dot blot检测TET2蛋白的表达水平和羟甲基化(5hmC)水平。DNA测序结果表明,TET2-gRNA-2靶位点附近分别发生2和28 bp的缺失突变,引起TET2蛋白移码突变,将该细胞株命名为DT-6。DT-6细胞传代15次以上,细胞形态稳定。Western blot结果显示,与DF-1细胞相比,DT-6细胞几乎不表达TET2蛋白。Dot blot结果显示,与DF-1细胞相比,DT-6细胞全基因组羟甲基化水平显著降低。利用CRISPR-Cas9技术成功构建了敲除TET2基因的鸡胚成纤维细胞系,为研究鸡TET2及其介导羟甲基化修饰参与调控天然免疫反应的途径和分子机制奠定基础。In order to studying the role of tet methylcytosine dioxygenase 2(TET2)and its mediated hydroxymethylation in regulating innate immune responses,CRISPR-Cas9 mediated gene editing systems were used to construct a TET2 knockout chicken embryonic fibroblast cell line.4 pairs of single guide RNAs(sgRNAs)were designed for targeting the conserved CDS sequences of the two RNA spliced isoforms of chicken TET2 gene.The plasmids expressing sgRNA was constructed,and were co-transfected with Cas9-T2A-GFP into DF-1 cells.DF-1 cells were transfected with a higher cleavage activity sgRNA.Then,the cells expressing GFP fluorescent were sorted by flow cytometry,and cultured and screened by limiting dilution method.The positive monoclonal cells were identified by DNA sequencing.When large numbers of the monoclonal knockout cells were genearated,the TET2 protein expression was detected by western blot and the level of hydroxymethylation(5hmC)was detected by dot blot.The results of DNA sequence showed that 2 and 28 bp deletion were detected in TET2 gene at position of the target site of TET2-gRNA-2.Both 2 deletions caused a reading frame shift and a premature stop codon.The clonal knockout cells were named DT-6.DT-6 cells were passaged for more than 15 times,the cell morphology was stable and the TET2 protein was not expressed.Compared with the DF-1 cells,the hydroxymethylation level of the cells was significantly reduced in DT-6 cells.In this study,the TET2 gene knockout chicken embryo fibroblast cell line was successfully constructed by applying CRISPR-Cas9 technology,which would provide a foundation for the further study of TET2 and its hydroxymethylation-mediated pathways and molecular mechanism in regulating innate immune response.

关 键 词：鸡 TET2 CRISPR-Cas9 TET2基因敲除 鸡胚成纤维细胞 

分 类 号：S831.2[农业科学—畜牧学]