【摘要】 目的观察构建的DNA载体介导的小干扰RNA Tet-on基因表达系统的调控效应。方法构建DNA载体介导的小干扰RNA(siRNA)Tet-on表达载体,pDIRS4.1 siRNA。以增强绿色荧光蛋白(EGFP)基因和白细胞介素6(IL-6)基因为目的基因,应用脂质体介导的瞬时转染方法,分别将pDIRS4.1/EGFP siRNA载体和p EGFP-N3质粒、pDIRS4.1IL-6siRNA载体和p IRESIL-6质粒共转染人成骨肉瘤细胞,加入系列浓度的四环素(Dox)后,观察其诱导效应。结果 pDIRS4.1 siRNA在人成骨肉瘤细胞中能够高效转染;随着Dox浓度升高,pDIRS4.1 siRNA载体在人成骨肉瘤细胞中对目的基因的沉默作用有较好的调控性,且目的基因的表达与Dox具有严格的剂量依赖关系,高浓度Dox时,IL-6表达水平降低约12倍。结论该研究构建的以DNA载体介导的能够高效调控siRNA沉默基因效应的Tet-On基因表达系统,能够促进其在更多模型系统中进行基因功能研究的应用。更多还原

【Abstract】 Objective To detect the regulatory effects of the established DNA vector-mediated Doxycycline-inducible siRNA system. Methods A DNA vector-mediated Doxycycline-inducible siRNA system, pDIRS4.1, was established. The regulatory effects of pDIRS4.1 were investigated by detecting the expression changes of enhanced green fluorescent protein(EGFP) and interleukin 6(IL-6) after pDIRS4.1/EGFP siRNA, p EGFP-N3 plasmid,pDIRS4.1/IL-6 siRNA and p IRES IL-6 plasmid had been cotransfected into U-2OS Tet-On cells respectively which were added with serial concentrations of Doxycycline(Dox). Results The results showed that a high transfection efficiency(70% ~80%) was obtained in the U-2OS Tet-On cells with pDIRS4.1 siRNA. pDIRS4.1 siRNA showed tight gene regulatory effects in the U-2OS Tet-On cells with the increase of Dox concentrations. Furthermore, the expressions of EGFP and IL-6 mediated by pDIRS4.1/EGFP siRNA and pDIRS4.1/IL-6 siRNA respectively had dose-dependent effect with the concentration of Dox. When the concentration of Dox was high, the expression level of IL-6 decreased 12 times. Conclusions A Dox-inducible siRNA expression system has been established through a non-viral mammalian expression vector. Tightly-regulated inducible siRNA expression systems of this type will likely have wide application in exploring gene functions in numerous model systems.更多还原