摘要
为通过慢病毒感染细胞构建稳定表达CRISPR/Cas9的293T细胞系,实现高效率基因编辑,PCR扩增Cas9基因片段,酶切后克隆到慢病毒载体pLent-EF1a-MF-CMV-GFP-P2A-Puro上,将该质粒与慢病毒包装质粒psPAX2、pMD2.G共感染293FT细胞,48 h后收集病毒并浓缩纯化测定其病毒滴度,最后用纯化后的病毒感染293T细胞,通过抗性和荧光标记筛选得到稳定表达Cas9的细胞系,并对单克隆细胞进行DNA、RNA特异性和反转录验证。针对ABCG4基因设计sgRNA并构建重组载体感染单克隆细胞,通过DNA特异性和T7E1酶切验证基因敲除效率。结果表明:成功构建了Cas9-pLent慢病毒载体,用构建成功的慢病毒载体对293FT细胞进行慢病毒包装(三质粒系统)并对包装成功的病毒进行了浓缩与纯化,最后通过病毒感染293T细胞,采用荧光观察的方式测定其病毒滴度均在106 TU·mL-1之上。用纯化后的病毒感染293T细胞并通过加药(Puro)进行抗性筛选,筛选出3株能稳定表达Cas9的293T细胞系,目的基因ABCG4的基因编辑功能验证表明其中1株T7E1酶切掉带明显,sgRNA靶向敲除效率较高,该株命名为Cas9-293T-10A。该研究建立了稳定表达Cas9的293T细胞系,可用于目的基因的高效敲除。
To establish the 293 T cell lines with CRISPR/Cas9 gene by lentiviral vector and further to realize the high-efficiency gene editing, Cas9 gene was amplified by PCR technique and inserted into the pLent-EF1 a-MF-CMV-GFP-P2 A-Puro vector. Monolayer of 293 FT cells were cotransfected with three plasmids psPAX2, pMD2.G and Cas9 in pLent-EF1 a-MF-CMV-GFP-P2 A-Puro. The recombinant lentivirus expressing Cas9 was harvested and the titer of virus was calculated by fluorescence microscope 48 h later. The purified lentivirus was used to infect 293 T cell, the transduced cells were selected with puromycin and single cell colonies were isolated and verified for DNA, RNA and cDNA. Designed sgRNA for ABCG4 gene and constructed recombinant vector to infect monoclonal cells, gene knockout efficiency was measured by DNA specificity and T7 E1 digestion. As results, DNA sequencing verified that Cas9-pLent was constructed successfully, and the titer of recombinant lentivirus was greater than 106 TU·mL-1. Three cell lines expressing Cas9 stably were obtained, one of which(designated Cas9-293 T-10 A) the targeted gene was knocked out successfully by T7 E1 digestion. In conclusion, 293 T cells stably expressing Cas9 was successfully constructed, which could be a valuable tool for gene editing.
作者
覃鸿妮
谢钰珍
杨晨晨
张勇
QIN Hongni;XIE Yuzhen;YANG Chenchen;ZHANG Yong(Research and Development Center of High-Throughput Gene Sequencing Engineering Technology,Suzhou Industrial Park Institute of Services Outsourcing,Suzhou 215123,China;Geneuuiz Biological Technology(Suzhou)Cornpany Limited,Suzhou 215123,China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2020年第4期29-35,45,共8页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
江苏高校自然科学研究面上项目(18KJD180005)
江苏省高职院校教师专业带头人高端研修项目(2019GRFX093)。
