| 【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. |
