利用CRISPR/Cas9技术构建PPARγ基因敲除的IPEC-J2细胞系  

Construction of IPEC-J2 Cell Lines with PPARγGene Knockout Mediated by CRISPR/Cas9 Technology

作　　者：张家翔 韩佃刚 师亚玲 毛晓月 赵凯薇 杜萱 信吉阁 ZHANG Jiaxiang;HAN Diangang;SHI Yaling;MAO Xiaoyue;ZHAO Kaiwei;DU Xuan;XIN Jige(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;Technology Center of Kunming Customs,Kunming 650200,China)

机构地区：[1]云南农业大学动物医学院,昆明650201 [2]昆明海关技术中心,昆明650200

出　　处：《中国畜牧兽医》2023年第7期2670-2677,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31960658、31360532);云南省自然科学基金项目(2013FB041);兴滇英才支持计划“青年人才”专项(YNWR-QNBJ-2020-154)。

摘　　要：【目的】利用CRISPR/Cas9技术敲除猪小肠上皮细胞(IPEC-J2)的过氧化物酶体增殖激活受体γ(PPARγ)基因,进而在细胞水平上研究PPARγ基因与肠道炎症反应相关的生物学机制。【方法】根据猪PPARγ基因序列(GenBank登录号:NM_214379),在第2外显子区域设计2对sgRNA序列,经退火形成DNA双链后连接至真核表达载体gRNA_GFP-T1;将构建成功的PPARγ正向打靶载体、反向打靶载体、Cas9-nickase质粒共转染至IPEC-J2细胞,经G418药物筛选获得单克隆细胞株,提取单克隆细胞株DNA后进行PCR扩增,经凝胶电泳鉴定后送测序;挑选基因敲除效果最佳的细胞株,利用免疫荧光法检测PPARγ蛋白表达情况。【结果】打靶载体测序结果显示,打靶序列正确连接;经转染、筛选后共获得48株单克隆细胞;测序结果显示,6株单克隆细胞出现双峰,分别为KO-15、KO-17、KO-21、KO-25、KO-32和KO-35;选择底峰较高的KO-25进行免疫荧光检测,结果显示PPARγ蛋白表达降低。【结论】本研究利用CRISPR/Cas9技术成功获得了PPARγ基因敲除的IPEC-J2细胞,为后续进一步研究PPARγ基因在炎症反应中的功能奠定了基础。【Objective】The purpose of this study was to use CRISPR/Cas9 technique to knock out the peroxisome proliferation-activated receptorγ(PPARγ)gene in porcine small intestinal epithelial cells(IPEC-J2),and to investigate the biological mechanism of PPARγgene associated with intestinal inflammation at the cellular level.【Method】According to porcine PPARγgene sequence(GenBank accession No.:NM_214379),two pairs of sgRNA sequences were designed in exon 2 region.After annealing,DNA double strand was formed and connected to the eukaryotic expression vector gRNA_GFP-T1.The constructed PPARγvector(forward target vector,reverse target vector)and Cas9-nickase plasmid were co-transfected into IPEC-J2 cells,after G418 drug screening,the monoclonal cell lines were sampled,DNA was extracted,PCR amplified,identified by gel electrophoresis and sequenced to select the cell lines with the best gene knockout effect,the expression of PPARγprotein was detected by immunofluorescence.【Result】The target vector sequencing results showed that the target sequences were correctly connected.A total of 48 monoclonal cells were obtained after transfection and screening.The sequencing results showed that 6 monoclonal cells showed double peaks,namely KO-15,KO-17,KO-21,KO-25,KO-32 and KO-35,respectively.The KO-25 with higher basal peak was selected for immunofluorescence detection,and the results showed that the expression of PPARγprotein decreased.【Conclusion】In this study,IPEC-J2 cells with PPARγgene knockout were successfully obtained using CRISPR/Cas9 technology,which laid the foundation for further research on the function of PPARγgene in inflammatory response.

关 键 词：CRISPR/Cas9 过氧化物酶体增殖激活受体γ(PPARγ) IPEC-J2细胞 基因敲除 

分 类 号：Q78[生物学—分子生物学]