CRISPR/Cas9介导的HO-1基因敲除细胞系的建立及其应用  

Establishment of CRISPR/Cas9-mediated HO-1 knockout cell line andits applications

作　　者：宋圣哲 罗通旺 沈灵俊 王书杰 郁孝强 宋厚辉 邵春艳 SONG Shengzhe;LUO Tongwang;SHEN Lingjun;WANG Shujie;YU Xiaoqiang;SONG Houhui;SHAO Chunyan(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Laboratory for Animal Health Inspection/Internet Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院,浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程实验室/动物医学与健康管理浙江省国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300

出　　处：《中国兽医学报》2024年第11期2463-2469,共7页Chinese Journal of Veterinary Science

基　　金：浙江农林大学科研发展基金人才启动基金资助项目(2020FR045);浙江省大学生科技创新活动计划暨新苗人才计划资助项目(2023R412021)

摘　　要：利用CRISPR/Cas9系统构建HO-1基因敲除的猪肾细胞系(PK-15),为探究HO-1在镉诱导的PK-15细胞铁死亡中的作用及分子机制提供试验材料。设计并合成3条针对HO-1基因的sgRNA,分别连接到慢病毒载体Lenti CRISPRv2上;将构建好的慢病毒质粒转染至人胚胎肾细胞(HEK293FT),获得重组慢病毒并感染PK-15细胞;经嘌呤霉素筛选和有限稀释法获得HO-1敲除的单克隆细胞系,测序及Western blot验证HO-1基因的敲除情况。用10μmol/L氯化镉(CdCl_(2))处理构建好的HO-1基因敲除的细胞,通过CCK-8法检测细胞活力,利用相差显微镜观察细胞形态,采用荧光探针法检测细胞中亚铁离子(Fe^(2+))的含量。结果显示,sgRNA2编辑效率最高,获得的5株敲除细胞HO-1基因均发生了碱基插入或缺失且引起了目的基因的移码,Western blot结果显示未检测到HO-1蛋白表达,表明成功构建了敲除HO-1基因的PK-15细胞系(PK-15^(HO-1 KO))。CdCl_(2)处理后,与对照细胞相比,PK-15^(HO-1KO)细胞活力显著升高,Fe^(2+)含量显著下降,表明HO-1基因敲除后可以显著缓解镉处理引起的铁代谢异常。综上所述,本研究利用CRISPR/Cas9基因编辑技术成功构建了敲除HO-1基因的PK-15细胞系,并证实HO-1在镉处理诱导PK-15细胞铁代谢异常中发挥着重要作用,为后续进一步研究HO-1在镉暴露致PK-15细胞铁死亡的调控机制奠定了基础。Porcine kidney cell line(PK-15)with HO-1 gene knockout was constructed by CRISPR/Cas9 system to provide experimental materials for exploring the role and molecular mechanism of HO-1 in cadmium-induced ferroptosis in PK-15 cells.Three sgRNA targeting HO-1 gene were designed,synthesized and ligated to lentiviral vector Lenti CRISPRv2.The constructed lentiviral plasmid was transfected into human embryonic cells(HEK293FT)to obtain recombinant lentivirus,which was used to infect PK-15 cells.The monoclonal cell line with HO-1 knockout was obtained by puromycin screening and limited dilution method.The knockout effect was analyzed by sequencing and Western blot detection.The HO-1 gene knockout cells were treated with 10μmol/L cadmium chloride(CdCl_(2)).The cell viability was detected by CCK-8 method,the cell morphology was observed by phase contrast microscope,and the content of ferrous ion(Fe^(2+))was detected by fluorescence probe.The results showed that sgRNA2 possessed the highest editing efficiency.The base insertion or deletion of HO-1 gene and the frameshift of the target gene occurred in all five knockout cells.Western blot results showed that no expression of HO-1 protein was detected,indicating that the PK-15 cell line with HO-1 gene knockout was successfully constructed.After CdCl_(2)treatment,compared with the control cells,the cell viability was significantly increased and the Fe^(2+)content was significantly decreased in PK-15 cells with HO-1 gene deletion,indicating that HO-1 gene knockout could significantly alleviate the abnormal iron metabolism caused by cadmium treatment.In conclusion,this study successfully constructed a PK-15 cell line knocking out HO-1 gene by using CRISPR/Cas9 gene editing technique,and confirmed that HO-1 plays an important role in iron metabolism abnormality in PK-15 cells induced by cadmium treatment,which lays a foundation for further study on the regulatory mechanism of HO-1 in ferroptosis in PK-15 cells induced by cadmium exposure.

关 键 词：HO-1 PK-15 基因敲除 CRISPR/Cas9 镉 

分 类 号：S852.3[农业科学—基础兽医学]