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目的:探索体内转录的短多聚腺苷酸[poly(A)]对外源基因mRNA的表达及出核转运的影响。方法:构建依赖于H1启动子体内转录的poly(A)载体,用脂质体转染方法将其与外源绿色荧光蛋白(GFP)基因表达质粒导入MCF-7细胞,采用qPCR和Western印迹分别检测GFP在mRNA和蛋白水平的表达情况,并通过体外实验检测体内转录的poly(A)对GFP mRNA的加尾影响;采用qPCR法考察16种内源基因的mRNA水平;将其与p53表达质粒共转染MCF-7细胞后,采用MTT法检测细胞增殖情况。结果:在MCF-7细胞中,依赖于H1启动子转录的poly(A)能够加速外源GFP mRNA的poly(A)加尾,促进其从细胞核向细胞质输出,从而提高12 h内GFP的表达量,对内源基因mRNA水平没有影响;它还能加速外源p53基因的表达。结论:建立了通过体内转录poly(A)从而加速外源基因表达的策略,可能对基因治疗或快速研究某个特异基因的功能具有重要的应用价值。
OBJECTIVE: To explore the effect of short poly-A [poly (A)] transcribed in vivo on the expression of exogenous gene mRNA and its nuclear export. METHODS: A poly (A) vector dependent on H1 promoter transcription in vivo was constructed and transfected into MCF-7 cells by lipofection method. The expression of GFP gene was detected by qPCR and Western blot The expression of GFP at mRNA and protein level was detected and the in vivo effect of poly (A) transfection on GFP mRNA was detected by qPCR method. The mRNA levels of 16 endogenous genes were detected by qPCR. After cotransfecting MCF-7 cells, MTT assay was used to detect cell proliferation. Results: In MCF-7 cells, poly (A) dependent on the H1 promoter transcription accelerated the poly (A) tail of exogenous GFP mRNA and promoted its export from the nucleus to the cytoplasm, thereby increasing the expression of GFP within 12 h Amount of endogenous gene mRNA level has no effect; it can also accelerate the exogenous p53 gene expression. CONCLUSIONS: Strategies have been established to accelerate exogenous gene expression by in vivo transcription of poly (A), which may have important implications for gene therapy or for rapid study of the function of a specific gene.