| 李晓娇 1，朱新宇 1，邹 娴 2，严 霞 2，何燕华 2，罗成龙 2.慢病毒介导稳定表达 Cas9 蛋白的鸡成纤维细胞系构建及其活性验证[J].广东农业科学,2023,50(2):125-135 |
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慢病毒介导稳定表达 Cas9 蛋白的鸡成纤维细胞系构建及其活性验证 |
| Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
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| DOI：10.16768/j.issn.1004-874X.2023.02.014 |
| 中文关键词: 稳定表达 慢病毒 CRISPR/Cas9 SSA 修复 载体构建 DF-1 |
| 英文关键词: stable expression lentivirus CRISPR/Cas9 SSA repair vector construction DF-1 |
| 基金项目: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） |
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| 中文摘要: |
| 【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-Hind Ⅲ，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。 |
| 英文摘要: |
| 【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-Hind Ⅲ to 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 established, which could provide basic materials for the subsequent research on chicken functional genes on the DF-1 cell line of stably expressing Cas9 protein. |
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