LSL-Cas9小鼠永生化胚胎成纤维细胞系的构建及应用  

Establishment and Application of Immortalized Cell Line of Mouse Embryonic Fibroblasts Derived from LSL-Cas9 Mice

作　　者：张言 包明月 韩冰 田梦园 杨亚娜 冯晨昱 李星[1] 张媛[1] ZHANG Yan;BAO Mingyue;HAN Bing;TIAN Mengyuan;YANG Yana;FENG Chenyu;LI Xing;ZHANG Yuan(College of Life Sciences,Shaanxi Normal University,Xi’an 710119,China)

机构地区：[1]陕西师范大学生命科学学院,西安710119

出　　处：《中国生物工程杂志》2024年第9期42-52,共11页China Biotechnology

基　　金：国家自然科学基金(9226811882271199)资助项目。

摘　　要：目的:利用猿猴病毒40(Simian virus 40,SV40)Large T(LT)抗原慢病毒转染含有LoxP-Stop-LoxP(LSL)元件的Cas9转基因小鼠(LSL-Cas9)胚胎成纤维细胞(mouse embryonic fibroblast,MEF),建立永生化LSL-Cas9小鼠MEF系并用于sgRNA剪切效率的高效验证。克服由于Cas9序列过大而引起的转染效率低的问题,构建一种用于sgRNA剪切效率验证的经济、便捷、高效工具。方法:以LSL-Cas9胎鼠为实验动物,分离其MEF并感染SV40 LT慢病毒;利用潮霉素筛选成功转化的细胞;通过免疫荧光检测成纤维细胞标志物表达和细胞衰老情况,通过聚合酶链反应(polymerase chain reaction,PCR)鉴定SV40 LT抗原基因在MEF中的整合情况,建立永生化无限增殖细胞系;以IL10Rα基因为例验证该系统的可行性:设计4种sgRNA,利用含有PU6-sgRNA串联PCSP-Cre基因表达盒的质粒转化永生化LSL-Cas9小鼠MEF系;通过T7内切核酸酶I(T7 endonuclease I,T7EI)法评估sgRNA的剪切效率,挑选靶向性最优的sgRNA。结果:经潮霉素筛选的阳性细胞扩大培养并稳定传代至60代,具备永生化无限增殖细胞系的特点;经PCR鉴定,SV40 LT抗原基因已稳定整合至MEF基因组中;T7EI酶切法证实sgRNA有效引导Cas9内切核酸酶实现了靶基因的切割,4种sgRNA的基因编辑效率分别为28.17%、45.87%、33.61%、38.62%。结论:SV40 LT抗原基因介导的永生化无限增殖LSL-Cas9 MEF系构建成功,可实现sgRNA引导Cas9剪切效率的高效快速验证。Objective:To facilitate the detection of sgRNA splicing efficiency,lentiviral transfection of embryonic fibroblasts(MEF)from LoxP-Stop-LoxP(LSL)-controlled Cas9 transgene-carrying(LSL-Cas9)mice with simian virus 40(SV40)large T(LT)antigen was utilized to establish an immortalized LSL-Cas9 mouse MEF line.By transforming the plasmid carrying the sgRNA expression cassette tandem with the constitutive promoter-driven Cre expression cassette into the cell line,regulatory expression of Cas9 can be realized.This system overcomes the problem of low transfection efficiency caused by the large Cas9 sequence,and provides an economical,convenient and efficient tool to verify the sgRNA shearing efficiency.Methods:LSL-Cas9 fetal mice were used as experimental animals,and their MEF cells were isolated and infected with SV40 LT lentivirus;hygromycin was used to screen successfully transformed cells.Detection of fibroblast marker expression and cellular senescence by immunofluorescence and the integration of SV40 LT antigen genes in MEF was identified by polymerase chain reaction(PCR),and a cell line with unlimited proliferation of immortalization was established.The IL10Rαwas used as a representative gene to verify the feasibility of this system.Four sgRNAs were designed and the immortalized MEF cell line from LSL-Cas9 mice was transformed using a plasmid containing a PU6-sgRNA wtih a tandem PCSP-Cre gene expression cassette.The sgRNA splicing efficiency was evaluated by the T7 Endonuclease I(T7EI)method,and the sgRNA with optimal targeting was selected.Results:Positive cells screened with hygromycin were expanded and stably passed to 60 generations,exhibiting the characteristics of immortalized,infinitely proliferating cell lines.The result of PCR identification showed that the SV40 LT antigen gene was stably integrated into the MEF genome.Conclusions:The SV40 LT antigen gene-mediated immortalized infinitely proliferating LSL-Cas9 MEF cell line was successfully established for efficient and rapid validation of sgRNA-guided Cas

关 键 词：LSL-Cas9转基因小鼠 永生化细胞系 SV40 LT抗原 T7EI 

分 类 号：Q291[生物学—细胞生物学]