基于胞嘧啶碱基编辑器构建ESM1基因敲除Hela细胞系  

Construction of ESM1 Gene Knockout Hela Cell Line Based on Cytosine Base Editor

作　　者：汪悦 郭颖 庞娅璇 崔堉熙 麻丽霞 WANG Yue;GUO Ying;PANG Yaxuan;CUI Yuxi;MA Lixia(Central Laboratory,Changzhi Medical College)

机构地区：[1]长治医学院中心实验室,山西长治046000

出　　处：《长治医学院学报》2024年第5期323-328,共6页Journal of Changzhi Medical College

基　　金：山西省基础研究计划(自由探索类)(202303021212231);长治医学院博士科研启动基金(202213);山西省高等学校科技创新计划项目(2023L210);大学生创新创业训练计划项目(20230862)。

摘　　要：目的:基于胞嘧啶碱基编辑器(CBE)提前引入终止密码子构建内皮细胞特异性分子1(ESM1)基因敲除Hela细胞系。方法:根据NCBI数据库ESM1序列信息(Gene ID:11082),利用在线软件Benchling在ESM1基因1号外显子上设计2个向导sgRNA,构建对应的pX330-ESM1-sgRNA表达载体。将BE4max、ACBE-ASR、pX330-ESM1-sgRNA 3个质粒共转染Hela细胞,用嘌呤霉素富集筛选CBE工作阳性细胞。PCR扩增含靶点的序列并进行Sanger测序,比较2个sgRNA的效率。通过有限稀释法筛选单克隆细胞,扩大培养后进行测序及Western Blot检测。结果:测序结果显示pX330-ESM1-sgRNA表达质粒构建成功,sgRNA2的编辑效率为66%,远高于sgRNA1的编辑效率;10组细胞单克隆中有6组细胞的靶点进行了双等位基因精确编辑,成功提前引入了终止密码子;Western Blot结果显示ESM1被成功提前终止翻译,达到基因敲除的目的。结论:基于胞嘧啶碱基编辑器提前引入终止密码子的策略成功构建了ESM1基因敲除Hela细胞系,为后续研究ESM1在宫颈癌中的作用机制提供了实验材料,奠定了基础。Objective:To establish an ESM1 gene knockout Hela cell line by introducing a stop codon in advance based on Cytosine Base Editor(CBE).Methods:Based on ESM1 sequence information from the NCBI database(Gene ID:11082),two guide sgRNAs were designed on exon 1 of ESM1 gene using online software Benchling,and the corresponding pX330-ESM1-sgRNA expression vector was constructed.BE4max,ACBE-ASR,and pX330-ESM1-sgRNA plasmids were co-transfected into Hela cells,and CBE-positive cells were enriched by puromycin selection.The target sequence was amplified by PCR and subjected to Sanger sequencing to compare the efficiency of the two sgRNAs.Single clone cells were selected through limiting dilution and sequencing and Western Blot were performed after expansion.Results:Sequencing confirmed that the successful construction of pX330-ESM1-sgRNA expression plasmid and an editing efficiency of 66%for sgRNA2,which was significantly higher than sgRNA1.Out of 10 cell clones,6 clones exhibited precise bi-allelic gene editing at the target site,resulting in the successful introduction of a stop codon.Western Blot analysis demonstrated the successful premature termination of ESM1 gene translation,which achieved the goal of gene knockout.Conclusion:The strategy of introducing a stop codon in advance based on Cytosine Base Editor(CBE)successfully constructed an ESM1 gene knockout Hela cell line,providing valuable experimental materials for studying the mechanism of ESM1 gene in cervical cancer.

关 键 词：胞嘧啶碱基编辑器 ESM1 基因敲除 

分 类 号：Q291[生物学—细胞生物学]