1.Construction of EZH2 gene site-directed knock-in Hut78 cells by CRISPR/Cas9 system
Zhuolin LU ; Xianjia XIONG ; Yundan WU ; Hui ZHOU ; Jun JIA ; Shuanglin WANG ; Lili WU ; Yijie LIU ; Yang QIAO ; Bing YANG ; Xiujuan ZHAO ; Qingsong WANG ; Chunyong HAN ; Ling ZHANG ; Yan SUN
Tianjin Medical Journal 2017;45(5):449-453
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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