【摘要】 目的:建立荧光显微镜快速筛选对血小板生成素受体(Mpl)基因RNA干扰片段的方法。方法:通过体外重组技术将Mpl与绿色荧光蛋白(GFP)构建成融合基因,克隆进pDs载体中产生pDs-Mpl-GFP载体,将人U6启动子及5个Mpl shRNA发夹结构(shMpl-A,shMpl-B,shMpl-C,shMpl-D,shMpl-E)分别克隆至pDs-Mpl-GFP真核载体中,构建出针对Mpl 5个不同位点的RNA干扰载体,然后将获得的上述干扰载体通过脂质体方法转染至293T细胞中,用荧光显微镜观察293T细胞的荧光发光强度及荧光细胞数,判定构建的RNA干扰载体对靶基因RNA的干扰效果。结果:构建的5个干扰载体都能对转染的293T细胞中的Mpl-GFP融合基因进行有效的基因敲减作用,在pDs-Mpl-GFP-TK-U6-shMpl-A、pDs-Mpl-GFP-TK-U6-shMpl-B与pDs-Mpl-GFP-TK-U6-shMpl-E的干扰作用下,293T细胞中的荧光强度及发荧光细胞数目变得更少和更弱,干扰效果较好,而其它两个效果较差;在这三个较好的干扰载体中,pDs-Mpl-GFP-TK-U6-shMpl-A的干扰效果最好。结论:成功构建Mpl基因的真核RNA干扰载体,并筛选出有效的RNA干扰片段。更多还原

【Abstract】 Objective: To establish a method for selecting effective Mpl-specific shRNA with fluorescence microscopy quickly.Methods: Mpl and green fluorescent protein(GFP) fused genes were constructed with recombination technique in vitro.Mpl-GFP fused genes was cloned into pDs vector resulting in pDs-Mpl-GFP.Human U6 promoter and five Mpl-specific shRNA(shMpl-A,B,C,D,E) were cloned into pDs-Mpl-GFP vector separately to obtain five RNA interference vectors aiming at different sites of Mpl gene.Then,vectors were transfected into 293T cells with liposome method.Take advantage of luminescence characteristic of GFP in these vectors,fluorescence intensity and the amount of fluorescent cells were detected under fluorescence microscopy to judge the effects of the shRNA.Results: The results showed that all the five vectors had knocking down effects on Mpl-GFP fusion protein in 293T cells,and fluorescence intensity and amount of fluorescent cells interfered by shMpl-A,B,E were less and weaker than those interfered by others.The effects of others were poor.The effect of shMpl-A was the best in them.Conclusions: The Mpl-specific shRNA interference vectors are successfully constructed and effective interference fragment is screened under fluorescence microscopy.更多还原