摘要
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） transcribed in vivo on exogenous gene mRNA nuclear export and expression. Methods： Plasmids were constructed to transcribe short poly（A） RNA in vivo regulated by H1 RNA polymerase Ⅲ promoter, and were co-transfected with the green fluorescent protein（GFP） gene or p53 vector into MCF-7 cells by using lipofectamine. The mRNA and protein expression levels of GFP were detected by qPCR and Western blot respectively, and the GFP mRNA fragments were tested by processing mRNA 3＇ ends in vitro. The mRNA level of selected 16 endogenous genes were examined by qPCR. MCF-7 cells containing transcribed short poly（A） and p53 vector were detected by MTT assay to analyze their proliferation. Results： Short poly（A） transcribed by H1 promoter in vivo could accelerate the formation of 3＇-poly（A） tail of exogenous GFP mRNA and speed up mRNA export from the nucleus to the cytoplasm in MCF-7 cells, thus it increased the GFP protein level in 12 h, however, it had no effect on endogenous genes expression; it also could accelerate exogenous p53 gene expression. Conclusion： By using the strategy of transcription of poly（A） in vivo, we could accelerate exogenous gene expression in cells which may be very useful in gene therapy or in fast exploring a specific gene function.
出处
《生物技术通讯》
CAS
2012年第3期318-323,共6页
Letters in Biotechnology
基金
国家重点基础研究发展计划(2011CB811300)
关键词
多聚腺苷酸
体内转录
外源基因
表达
出核转运
加速
poly（A）
transcribed in vivo
exogenous gene
expression
nuclear export
accelerate
