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目的构建表达下游调控元件的拮抗分子(DREAM)基因序列特异性的短发卡RNA(shRNA)质粒载体,检测其对C6细胞DREAM基因的干扰作用,为研究DREAM基因功能提供有力工具。方法为了构建pRNAT-U6.1/Neo载体介导DREAM shRNA表达的质粒,针对DREAM的19碱基大小的片段,分别设计3对寡核苷酸和1条非特异性阴性对照序列,形成双链后将其依次连入带有U6启动子的pRNAT-U6.1/Neo载体(命名为pRNAT-DREAM),两对DNA双链连接后形成中间由9个碱基序列间隔的反向互补序列,构建成能产生DREAM shRNA的质粒。用构建成功的4条质粒转染C6细胞,分别采用Realtime PCR和蛋白印迹检测其DREAM基因的mRNA和蛋白表达。结果酶切、连接后构建成的质粒,经酶切与测序证实构建成功,无任何碱基突变。在培养的神经胶质细胞中转染率为85%左右,pRNAT-DREAM-Ⅱ和pRNAT-DREAM-Ⅲ显著抑制DREAM基因的表达。结论成功构建了能表达DREAM shRNA的质粒载体pRNAT-DREAM-Ⅱ、pRNAT-DREAM-Ⅲ,此项研究结果为DREAM信号通路的深入研究奠定了基础。
Objective To construct short hairpin RNA (shRNA) plasmids expressing the downstream regulatory element of DREAM gene and to detect its interference with DREAM gene in C6 cells, so as to provide a powerful tool for studying the function of DREAM gene. Methods In order to construct pRNAT-U6.1 / Neo vector-mediated DREAM shRNA expression plasmid, we designed three pairs of oligonucleotides and one non-specific negative control sequence for the 19-base fragment of DREAM This was ligated into the pRNAT-U6.1 / Neo vector (named as pRNAT-DREAM) with the U6 promoter, and the two DNA double strands were ligated to form a reverse complementary sequence with a 9-base sequence interval therebetween A plasmid that produces DREAM shRNA. C6 cells were transfected with the four constructed plasmids, and the mRNA and protein expression of DREAM gene were detected by Realtime PCR and Western blot, respectively. Results After digestion and ligation, the constructed plasmids were successfully constructed by enzyme digestion and sequencing without any base mutation. In cultured glial cells, the transfection efficiency was about 85%. PRNAT-DREAM-Ⅱ and pRNAT-DREAM-Ⅲ significantly inhibited the expression of DREAM gene. Conclusion Plasmid vectors pRNAT-DREAM-Ⅱ and pRNAT-DREAM-Ⅲ, which can express DREAM shRNA, were successfully constructed. The results of this study laid the foundation for the further study of DREAM signaling pathway.