原代小鼠卵巢上皮细胞TP53基因敲除及其生物学特征变化

目的 利用CRISPR/Cas9慢病毒载体系统建立小鼠原代卵巢上皮细胞TP53基因稳定敲除细胞系，分析细胞增殖、细胞周期、克隆形成以及细胞转移侵袭能力的变化。方法 构建LentiCRISPRv2-sgRNA TP53基因敲除质粒，用293FT细胞进行慢病毒包装，转导小鼠原代卵巢上皮细胞，嘌呤霉素筛选出稳定敲除细胞系，进行PCR、蛋白免疫印迹以及免疫荧光鉴定。细胞增殖、细胞周期变化、克隆形成、细胞迁移侵袭能力分别用MTT、流式细胞分析、单层培养以及Transwell小室进行测定。结果 TP53基因敲除小鼠原代卵巢上皮细胞中P53表达缺失；TP53敲除引起细胞迅速增殖，DNA合成加速，克隆形成以及迁移侵袭能力增强。结论 获得了原代小鼠卵巢上皮细胞TP53基因稳定敲除细胞系，细胞生物学特征明显改变。

P53 Knockout in Mouse Primary Ovarian Epithelial Cells and Biological Characteristic Changes

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．