利用CRISPR/Cas9系统构建Slc35c1敲除细胞株及其抗蓖麻毒素效应研究  

Construction of Slc35c1 gene knockout cell line by CRISPR/Cas9 system and its anti-ricin effect

作　　者：赵雨 翟雅洁 袁蜜蜜 邓亚楠 王勃 王玉霞 王籽橙 段小涛 ZHAO Yu;ZHAI Ya-jie;YUAN Mi-mi;DENG Ya-nan;WANG Bo;WANG Yu-xia;WANG Zi-cheng;DUAN Xiao-tao(Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;School of Pharmacy,Yantai University,Yantai,Shandong 264005,China;Fifth Medical Center of Chinese PLA General Hospital,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院毒物药物研究所,北京100850 [2]烟台大学药学院,山东烟台264005 [3]解放军总医院第五医学中心,北京100071

出　　处：《军事医学》2021年第11期823-827,共5页Military Medical Sciences

基　　金：国家自然科学基金面上项目(81673387)。

摘　　要：目的构建稳定敲除小鼠Slc35c1基因的单克隆细胞株,为研究Slc35c1基因抗蓖麻毒素毒性效应奠定实验基础。方法根据Slc35c1基因序列设计2对sgRNA插入载体lentiCRISPRv2中,构建lentiCRISPRv2-sgRNA质粒;慢病毒包装并感染NIH/3T3细胞,加入嘌呤霉素筛选阳性细胞;T7E1酶切鉴定sgRNA切割效率;利用有限稀释法筛选单克隆细胞并进行RT-qPCR检测及基因组PCR产物测序,确认Slc35c1敲除成功的细胞株;最后通过CCK-8试剂检测蓖麻毒素对野生型和Slc35c1敲除细胞对的抗毒效应差异。结果测序结果显示sgRNA成功插入载体质粒中,T7E1酶切结果表明两对sgRNA均可高效切割基因组DNA。RT-qPCR及基因组PCR产物测序鉴定出Slc35c1敲除的纯合子细胞株。CCK-8实验结果显示,与野生型细胞相比,敲除Slc35c1使毒素作用NIH/3T3细胞24 h的IC_(50)值由0.45×10^(-10)mol/L升至2.1×10^(-10)mol/L。结论利用CRISPR/Cas9系统靶向敲除了NIH/3T3细胞中的Slc35c1基因并筛选获得纯合子细胞株,该细胞株可用于抗蓖麻毒素效应的研究。Objective To construct a mouse Slc35c1 gene knockout NIH-3T3 cell line using the CRISPR/Cas9technique and to provide data for studying the anti-ricin toxicity of Slc35c1 gene.Methods Two pairs of small guide RNA targeting the Slc35c1 gene were designed and inserted into vector lentiCRISPRv2 before the recombinant plasmid lentiCRISPRv2-sgRNA was packaged by the lentiviral packaging system.Then,the NIH/3T3 cells were infected with the virus and screened by puromycin.The cleavage efficiency of sgRNA was identified by T7E1 digestion.Monoclonal cells were acquired with the limited dilution method,and the amplified monoclonal cells were detected by RT-qPCR and the genomic PCR products sequences so as to identify the monoclonal cell line with actual gene knockout.Finally,the anti-toxin effects of wild type and Slc35c1 knockout cell lines on ricin were compared by CCK-8 assay.Results The sequencing results confirmed the successful insertion of sgRNA into the plasmid vector.T7E1 digestion experiment showed that both pairs of sgRNA could efficiently cut the genomic DNA.A homozygous cell line with successful gene knockout was identified by RT-qPCR and genomic PCR product sequences.Further experiments showed that Slc35c1 knockout enhanced the ricin resistance of NIH/3T3 cells,and that the IC_(50)value of 24 h increased from 0.45×10^(-10)mol/L to 2.1×10^(-10)mol/L.Conclusion Slc35c1 gene in NIH/3T3 cells is knocked out by CRISPR/Cas9 system and a homozygous monoclonal cell line is successfully obtained,which can be used to study the anti-ricin effect.

关 键 词：CRISPR/Cas9系统 蓖麻毒素 Slc35c1基因 基因敲除 

分 类 号：R285[医药卫生—中药学]