摘要
【目的】筛选稳定表达Cas9蛋白的鸡成纤维细胞系(DF-1),并基于单链退火修复机制(Single Strand Annealing, SSA)的报告载体系统,检测DF-1细胞系中的Cas9核酸酶活性。【方法】将携带Cas9蛋白的慢病毒载体质粒与辅助质粒一起转染293T细胞包装慢病毒,收集慢病毒Cas9上清液感染DF-1细胞,经抗生素筛选得到稳定表达Cas9蛋白的DF-1细胞,分别用PCR和Western blot验证阳性DF-1细胞表达Cas9蛋白的情况;选择鸡卵清白蛋白(OVA)基因的sgRNA序列,将sgRNA退火产物克隆至pYP152构建sgRNA表达载体,并在sgRNA靶位点两端设计引物扩增OVA基因靶片段,将靶片段克隆至报告载体pCMV-SSA-mCherry-HindⅢ,以破坏mCherry蛋白的表达,之后再将sgRNA表达载体与SSA报告载体共同转染稳定表达Cas9蛋白的DF-1细胞,最后在荧光显微镜下分析不同稳转株对mCherry蛋白表达的修复情况。【结果】经抗生素筛选得到27株稳定表达Cas9蛋白的DF-1细胞系,采用PCR扩增稳转细胞基因组,结果显示这些细胞具有Cas9蛋白基因序列,Western blots试验结果显示上述稳转细胞株均表达Cas9蛋白;sgRNA表达载体与mCherry-SSA报告载体共转后,通过荧光显微镜观察发现筛选到的稳转细胞株均可恢复报告载体中mCherry蛋白的表达。【结论】成功构建了具有切割活性的稳定表达Cas9蛋白的DF-1细胞系,可为后续在稳定表达Cas9蛋白的DF-1细胞系上开展鸡相关功能基因研究提供基础材料。
【Objective】The chicken fibroblast cell line(DF-1)of stably expressing Cas9 protein was screened,and combined with the reporter vector system based on single strand annealing(SSA)repair mechanism,the Cas9 nuclease activity in the established DF-1 cell line was detected.【Method】The lentiviral vector plasmid carrying Cas9 protein was transfected into 293T cells packaging lentivirus together with the helper plasmid.DF-1 cells were infected with lentivirus Cas9 supernatant,and DF-1 cells with stable Cas9 protein expression were obtained through antibiotic screening.The expression of Cas9 protein in positive screened DF-1 cells was verified by polymerase chain reaction(PCR)and Western blot.The sgRNA sequence of chicken ovalbumin(OVA)gene was selected and and the annealing products were cloned into pYP152 to construct sgRNA expression vector.Primers were designed at both ends of the sgRNA target site to amplify the target fragment of OVA gene.The target fragment was cloned into mCherry-SSA reporter vector pCMV-SSA-mCherry-HindⅢto destroy the expression of mCherry protein.Then the sgRNA expression vector and mCherry-SSA reporter vector were co-transfected into DF-1 cells of stably expressing Cas9 protein.Finally,the repair of mCherry expression by different stable transformants was analyzed by fluorescence microscope.【Result】27 DF-1 cell lines of stably expressing Cas9 protein were obtained after antibiotic screening,cell genome was stably transfected by PCR amplification,and the results showed that these cells contained Cas9 protein sequences.The results of Western blot assay showed that all the stable transfection cell lines expressed Cas9 protein;after co-transformation of sgRNA expression vector and mCherry-SSA reporter vector,fluorescence microscope observation showed that all the selected stable transfection cell lines could restore the expression of mCherry protein in the report vector.【Conclusion】The DF-1 cell line of stably expressing Cas9 protein with cleavage activity was successfully establi
作者
李晓娇
朱新宇
邹娴
严霞
何燕华
罗成龙
LI Xiaojiao;ZHU Xinyu;ZOU Xian;YAN Xia;HE Yanhua;LUO Chenglong(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Institute of Animal Science,Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock Breeding/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research,Guangzhou 510640,China)
出处
《广东农业科学》
CAS
2023年第2期125-135,共11页
Guangdong Agricultural Sciences
基金
云浮市2021年省乡村振兴战略专项(2021020608)
广东省重点领域研发计划项目(2022B0202110002)
国家自然科学基金青年科学基金(32102539)
广东省农业科学院协同创新中心项目(XT202217)
国家肉鸡产业技术体系岗位科学家项目(CARS-41)。
