【摘要】 目的建立microRNA失功能(loss of function)研究模型,为功能研究提供实验材料。方法针对miR-483-5p成熟体序列设计含6个拷贝反义序列的DNA片段,称为"S-483",将其连接入含CAG启动子的pCAGGs载体中,构建pCAGGs-S-483重组质粒。利用脂质体技术瞬时转染Hep1-6细胞,经re-al-time PCR对成熟miR-483-5p含量进行检测。结果转染pCAGGs-S-483组miRNA-483含量(0.33±0.04)较转染空质粒组(1.00)明显下降(P<0.01),但对其它miRNAs无影响。结论 S-483海绵策略可有效"吸附"并降低miR-483-5p,成功构建了pCAGGs-S-483重组质粒,为其功能研究和miRNA敲减动物的建立提供了有益借鉴。更多还原

【Abstract】 Objective To study microRNA function in vitro,a genetic strategies is required to generate loss-of-function phenotype.Methods The recombinant plasmids which expressed "microRNA Sponges",a six copies antisense sequence to mature microRNA miR-483-5p,were constructed.The miR-483-5p sponges plasmids were transient transfected into mouse Hepa1-6 cells.Real-time PCR was performed to identify the amounts of mature miR-483-5p.Results The amount of mature miR-483-5p was knockdowned(0.33±0.04) by the microRNA sponges recombinant plasmids with control group as 1.00(P<0.01).Conclusion The microRNA sponges strategies to knockdown microRNA is effective in loss-of-function research.更多还原