利用改良的CRISPR/Cas9n double nick系统构建DPF2基因敲除的人胰腺癌PANC-1细胞株  

Construction of DPF2 Gene Knockout Human Pancreatic Cancer PANC-1 Cell Line Using Improved CRISPR/Cas9n Double Nick System

作　　者：黄卫东[1] 张文胜 陈一帆[1] 郭嘉义[3] HUANG Weidong;ZHANG Wensheng;CHEN Yifan;GUO Jiayi(School of Basic Medical Science,Ningxia Medical University,Yinchuan 750004,China;School of Medicine,Soochow University,Suzhou 215123,China;Medical Science and Technology Research Center,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院,银川750004 [2]苏州大学医学院,苏州215123 [3]宁夏医科大学医学科学技术研究中心,银川750004

出　　处：《宁夏医科大学学报》2023年第12期1189-1193,共5页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2018AAC03075);宁夏回族自治区重点研发计划项目(2022BEG03151)。

摘　　要：目的利用改良的CRISPR/Cas9n double nick系统构建DPF2基因敲除的PANC-1人胰腺癌细胞模型。方法设计两对靶向DPF2基因第四外显子上下游的单链向导RNA(single guide RNA,sgRNA),化学合成sgRNA寡核苷酸序列,并克隆至pGL3-U6-sgRNA-PGK-puromycin真核表达质粒中。将克隆正确的DPF2-sgRNA重组真核表达质粒与Cas9真核表达载体pST1374-N-NLS-flag-linker-Cas9共转染至PANC-1细胞中,通过嘌呤霉素和杀稻瘟菌素筛选阳性转染细胞,进一步通过有限稀释法筛选获得单克隆细胞株。提取细胞基因组DNA对敲除位点进行聚合酶链反应(PCR)鉴定和测序鉴定。Western blot检测细胞中DPF2蛋白表达情况。结果sgRNA成功插入pGL3-U6-sgRNA-PGK-puromycin中且序列正确。PCR扩增鉴定和测序结果表明,PANC-1细胞中一段包括完整第四外显子在内的长度为401 bp的DPF2基因片段被敲除。Western blot检测结果表明,DPF2基因敲除细胞中的DPF2蛋白表达缺失。结论通过改良的CRISPR/Cas9n double nick基因编辑系统成功构建DPF2基因敲除PANC-1稳定细胞株。Objective Construction of DPF2 knockout PANC-1 pancreatic cancer cell model with improved CRISPR/Cas9n double nick system.Methods The two pairs of single guided RNA(sgRNA)were designed for targeting the upstream and downstream of the DPF2 gene Exon 4.Oligonucleotide sequences of sgRNA were chemically synthesized and then cloned into the pGL3-U6-sgRNA-PGK-puromycin eukaryotic expression plasmid.pST1374-N-NLS-flag-linker-Cas9 plasmid DNA and DPF2-sgRNA plasmid DNA were co-transfected into PANC-1 cells.The positively transfected cells were screened by puromycin and blasticidin,and a monoclonal cell strain was obtained through limited dilution screening.The genomic DNA of the positive cells was extracted and knockout site of DPF2 gene was verified by PCR and sequencing.Total cellular proteins were extracted and the expression of DPF2 was analyzed by Western blot.Results sgRNA duplex was successfully inserted into pGL3-U6-sgRNA-PGK-puromycin correctly.The PCR amplification and sequencing results showed that a 401 bp fragment of the DPF2 gene was knocked out in PANC-1 cells,leading to loss of DPF2 protein expression in DPF2 gene knockout cells.Conclusion The DPF2 knockout PANC-1 stable cell line was successfully constructed by the CRISPR/Cas9n double nick gene editing system.

关 键 词：DPF2基因 CRISPR/Cas9n double nike系统 基因敲除 PANC-1细胞株 

分 类 号：R180.37[医药卫生—流行病学]