【摘要】 目的:应用CRISPR/Cas9技术构建Fbxw7表达抑制的Raw264.7稳定巨噬细胞株,并检测其生理功能。方法:转染Fbxw7_Cas9KO质粒到Raw264.7细胞,用含嘌呤霉素的培养基培养,挑取单克隆并筛选带荧光标记的细胞进一步培养;然后采用qRT-PCR和Western blot分别检测Fbxw7基因与蛋白表达水平;Confocal进一步检测Fbxw7蛋白表达分布;测序分析基因突变情况;CCK-8和流式细胞术分别检测Fbxw7基因敲低后Raw264.7细胞增殖能力与凋亡率。结果:Fbxw7敲低组的mRNA和蛋白表达水平较正常Raw264.7细胞显著降低,Confocal观察发现敲低组Raw264.7细胞中Fbxw7蛋白表达微弱;测序结果显示Fbxw7敲低组存在多位点突变;Fbxw7敲低后Raw264.7细胞增殖能力明显降低,凋亡率增加。结论:利用CRISPR/Cas9技术可以实现Fbxw7基因在Raw264.7细胞稳定低表达,所筛选出的稳定细胞株可用于后续细胞水平的实验研究。更多还原

【Abstract】 Objective:Establish a stable Fbxw7 knockdown expression cell line in Raw264.7 macrophage by using CRISPR/Cas9 program and array its associated hpenotype and function.Methods:Raw264.7 macrophage was transinfected with Fbxw7-Cas9 KO plasmid and cultured in DMEM medium that containing puromycin,and then single colony carried RFP fluorescence was sorted.The dynamics of mRNA and protein levels of Fbxw7 of Raw264.7 cell was detected by qRT-PCR and Western blot,respectively.Expression and distribution of Fbxw7 in Raw264.7 cell was observed under confocal microscope.Then the Fbxw7-mutated cell was cultured and genomic was extracted and sequenced.Finally,viability and apoptosis of Fbxw7-mutated Raw264.7 cells was assayed by CCK-8 kit and flow cytometry(FCM).Results:Fbxw7 expression levels of mRNA and protein were markedly decreased in Fbxw7 knock-down cells compared to the normal Raw264.7 macrophage.Confocal observation demonstrated that Fbxw7 expression was extremely weak in Fbxw7 knock-down Raw264.7 macrophages.Sequencing data revealed that there were many mutation sites in Fbxw7-Cas9 KO DNA.For Fbxw7 knock-down Raw264.7 cells,its viability was significantly decreased compared to normal cells,while its apoptosis was higher than normal Raw264.7 cell line.Conclusion:Fbxw7-Cas9 KO plasmid was successfully transinfected into Raw264.7 cells and decreased Fbxw7 expression level and the stable cell line was acquired.Fbxw7-mutated Raw264.7 cells presented reduced viability and increased apoptosis and may be provide as a new platform for the research of Fbxw7 function in Raw264.7 macrophages.更多还原