目的 利用CRISPR/Cas9系统构建定点敲入EZH2基因的Hut78细胞系.方法 构建EZH2表达载体pMD-18T-EZH2和单向导RNA(sgRNA)表达载体pSpCas9(BB)-2A-Puro-sgRNA,将两个载体共转染Hut78细胞,通过qPCR检测EZH2 mRNA的表达情况,通过Western Blot检测EZH2和H3K27me3蛋白的表达情况.结果 测序结果 显示,构建的pMD-18T-EZH2和pSpCas9(BB)-2A-Puro-sgRNA表达载体插入序列完全正确.将构建的两个质粒共转染Hut78细胞,qPCR结果 显示EZH2基因在RNA水平上表达显著上调;Western blot结果 显示EZH2和H3K27me3蛋白表达水平显著提高.结论 通过CRISPR/Cas9系统成功构建了定点敲入EZH2基因的Hut78细胞系.%Objective To construct the Hut78 cell line with EZH2 gene knocked into by CRISPR/Cas9 system. Methods The EZH2 expression vector pMD-18T-EZH2 with homologous arm and the sgRNA expression vector pSpCas9 (BB)-2A-Puro-sgRNA, which could cut the double stranded genomic DNA, were constructed, and the two vectors were co-transfected into Hut78 cells. Then the expression of EZH2 mRNA was detected by qPCR, and the expressions of EZH2 and H3K27me3 proteins were detected by Western blot assay. Results The pMD-18T-EZH2 and pSpCas9(BB)-2A-Puro-sgRNA recombinant vectors were confirmed by DNA sequencing. When Hut78 cells were transfected with the two recombinant plasmid, qPCR results showed that the expression of EZH2 mRNA was significantly increased, and Western blot analysis showed that the expressions of EZH2 and H3K27me3 proteins were significantly increased. Conclusion EZH2 gene is successfully knocked into Hut78 cells by CRISPR/Cas9 system.
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