原代小鼠卵巢上皮细胞TP53基因敲除及其生物学特征变化
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摘要
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Lenti CRISPRv2-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。
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 Lenti CRISPRv2-sgRNA TP53 gene knockout plasmid was constructed and packaged in 293 FT 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.
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 Lenti CRISPRv2-sgRNA TP53 gene knockout plasmid was constructed and packaged in 293 FT 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.
引文
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[2]Cong L,Zhang F.Genome engineering using CRISPR-Cas9system[J].Methods Mol Biol,2015,1239:197-217.
[3]Wang X,Huang X,Fang X,et al.,CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells[J].Mol Ther Nucleic Acids,2016,5:e388.
[4]Zhang F,Wen Y,Guo X.CRISPR/Cas9 for genome editing:progress,implications and challenges[J].Hum Mol Genet,2014,23(R1):R40-46.
[5]Herrero A B,Rojas E A,Misiewicz-Krzeminska I,et al.,Molecular Mechanisms of p53 Deregulation in Cancer:An Overview in Multiple Myeloma[J].Int J Mol Sci,2016,17(12):2-20.
[6]Hientz K,Mohr A,Bhakta-Guha D,et al.,The role of p53 in cancer drug resistance and targeted chemotherapy[J].Oncotarget,2017,8(5):8921-8946.
[7]Mantovani F,Walerych D,Sal G D,Targeting mutant p53 in cancer:a long road to precision therapy[J].FEBS J,2016,1742-4658.
[8]Ebata T,Hirata H,Kawauchi K.Functions of the Tumor Suppressors p53 and Rb in Actin Cytoskeleton Remodeling[J].Biomed Res Int,2016,2016:9231057.
[9]Zanjirband M,Edmondson R J,Lunec J.Pre-clinical efficacy and synergistic potential of the MDM2-p53 antagonists,Nutlin-3and RG7388,as single agents and in combined treatment with cisplatin in ovarian cancer[J].Oncotarget,2016,7(26):40115-40134.
[10]Cole A J,Dwight T,Gill A J,et al.Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing[J].Sci Rep,2016,6:26191.
[11]Jiang W,Bikard D,Cox D,et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J].Nat Biotechnol,2013,31(3):233-239.
[12]金镇,关婷,李守柔,野生型p53基因转染卵巢癌SKOV-3细胞对化疗药物顺铂敏感性的影响[J].中华医学遗传学杂志,2002,(3):40-42.
[13]Orsulic S,Li Y,Soslow R A,et al.Induction of ovarian cancer by defined multiple genetic changes in a mouse model system[J].Cancer Cell,2002,1(1):53-62.
[14]Wang S P,Wang W L,Chang Y L,et al.p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug[J].Nat Cell Biol,2009,11(6):694-704.
[15]Zhou F,Xu Y,Shi J,et al.Expression profile of E-cadherin,estrogen receptors,and P53 in early-onset gastric cancers[J].Cancer Med,2016,5(12):3403-3411.
[16]Castro Alves C,Rosivatz E,Schott C,et al.,Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin[J].J Pathol,2007,211(5):507-515.