关键词: CRISPR/Cas9, TP53, 基因敲除, 小鼠卵巢上皮细胞

Abstract: Objective To establish a TP53 gene knockout cell line using CRISPR/Cas9 system in mouse primary ovarian epithelial cells(MPOE cells), and study the effect of TP53 gene knock out on cell proliferation, cell cycle progression, colony formation,migration and invasion.Method The LentiCRISPRv2-sgRNA TP53 gene knockout plasmid was constructed and packaged in 293FT cell. The supernatant was collected，and infected the MPOE cells after filtered．The positive clones were selected by puromycin. PCR, Western blot and Immunofluorescence were used to detect TP53 knockout cell lines．Cell proliferation, cell cycle progression, colony formation, migration and invasion were examined by MTT, flow cytometry analysis, colony formation assay and transwell assay, respectively. Result TP53 knockout MPOE cell line was generated in which cell proliferation and colony formation were increased，DNA synthesis was elevated, moreover, cell migration and invasion were increased. Conclusion The TP53 gene stable knockout cell line of MPOE was successfully generated by CRISPR/Cas9 system and the biological characteristics changed obviously．

Key words: CRISPR/Cas9, TP53, gene knockout, mouse primary ovarian epithelial cells