西"农业学& Southwest China Journal of Ag/cultural Sciences 2020332 Vol.33No.2
246
文章编号：1001-4829(2020)2-0246-06DOI：10.16213/jkl.scjas.2020.2.005
覃鸿妮1，谢饪珍打陈罡2，密苗苗3，彭赛男3，张勇1*
(1•苏州工业园区服务外包职业学院&江苏苏州215123;2.金唯智生物科技有限公司&江苏苏州215123;3.斯澳生物科技(苏州)有限公司&江苏苏州215123)
摘要：【目的】通过慢病毒转染细胞构建稳定表达CRISPR/Lpt的293T和HT1080细胞系，实现高效率基因编辑。【方法】PCR 扩增At p t和LbCpfl基因片段，酶切后克隆到慢病毒载体pLent-TF1a-MF-TMV-GFP-P2A-Ture上，将该质粒与慢病毒包装质粒psPAX2、pMD2.G共转染293FT细胞，通过抗性和荧光标记筛选得到稳定表达LbCpf1和Aipf1的细胞系，并对单克隆细胞进行DNA、RNA特异性和反转录验证。用设计好的针对ABCG1基因的sgRN A转染单克隆细胞，通过DNA特异性和T7E1酶切验证基因敲除
效率。【结果】成功构建了Cpf1-pLent慢病毒载体，重组载体对293FT细胞进行慢病毒包装、浓缩与纯化后，病毒滴度均在106TU/mL之上。用纯化后的病毒感染293T和HT1080细胞#筛选出3株能稳定表Cpt的细胞系，目的ABCG1基因的基因编辑功能验证表明其中2株T7EI酶切掉带明显#p RN A靶向敲除效率较高，命名为APpf1D93T-7D和APpf1磷T10%002-3B$【结论】建立了稳定表达Cpfl的293T和HT1080细胞细胞系，可以用于目的基因的高效敲除。
关键词：CRISPR/Cpf1；慢病毒载体；细胞系(基因编辑
中图分类号:S154.32文献标识码:A
Construction of Cell Lines wits Stably Expressing Cpf1Gene
by Leetiviral Vector and ideetitcation of Functions
QIA Hong-nl1&XI Yu-zhen1&CHEN Gang2&MI Miao-miao3&PENG Sai-nan3&ZHANG Yong1*
(1.Suzhou Industrial Park Institute of Services Outsourcing,Jiangsu Suzhou215123,China, 2.Genewis Biological Technology Co.,Ltd.,
Jiangsu Suzhou215123,China；3.Siao Biotechnology(Suzhou)Co.,Ltd.,Jiangsu Suzhou215123,China)
Abstract:(Objective]This paper aimed to establish the293T and HT1080cell lines with CRISPR/Cpf1gene by lentiviral vector and further to realize the high-eXiciency gene editing.(Method]AsCpf1and LbCpf1gene was amplified by PCR technique,which was inserted into the pLent-TF1a-MF-TMV-GFP-P2A-Ture vector.Monolayer of293FT cells were cotransfected with three plasmids psPAX2,pMD2.G and Cpt in pLent-EF1a-MF-CMV-GFP-P2A-Pui/.The recombinant lentivirus expressing LbCptand AsCpf1was harvested,and the titer of virus was calculated by fluorescence microscope48hours later.The pu/fied lentivirus was used to infect293T cell and the transduced cells were selected with puromycin and single cell colonics were isolated and ve/fied for DNA,RNA and cDNA.Single cell colonics were transfected by sgRNA tai/eting the exons ABCG1CDS reyions to the functional domains of ABCG1genes,and gene knockout eXiciency was measured by DNA specificity and T7EI digestion.(Result]DNA sequnencing ve/fied that Cpf1-pLent was constructed successfully,and the titer of recombinant lentivirus was greater than106TU/mL.Three cell lines expressing Cpf1stably were obtained,two of which(AsCpf1-293TGD and AsCpf1-HT108002-3B)t he targeted gene was knocked out successfully by T7EI digestion.(Conclusion]293T and HT1080 cells stably expressing Cpf1was successfully constructed,which could be a valuable tool for gene editing.
收稿日期：2019-04-10
基金项目：江苏省高等学校自然科学研究面上项目(18KJ D180005)%江苏省高职院校教师专业带头人高端研究项目(2019GRFX093)；江苏省高等学校大学生创新创业训练计划项目(201814295006Y)
表达载体构建作者简介：覃鸿妮(1983-)J，湖北长阳人，博士，讲师，研究方向为分子生物学，*为通讯作者：张勇(1971-) &男，南京六合人，硕士&高级实验师&研究方向为有机化学’Key words：CRISPR/Cpf1；Lentiviral vector；Cell line；Gene
editing
【研究意义］慢病毒载体是一种强大、灵活的载体工具，可以在体外和体内将基因转移至分裂细胞和非分裂细胞。慢病毒载体因为其独有的特点，在近年引发了不同领域的关注，这些载体被大
豫公网安备41160202000603
站长QQ:729038198
关于我们
投诉建议