小鼠微RNA miR-21真核表达质粒的构建及其在小鼠肾小球系膜细胞中的表达

目的构建小鼠微RNA miR-21真核表达质粒,并在小鼠肾小球系膜细胞中表达。方法人工合成小鼠miR-21基因序列,构建miR-21真核表达质粒pGenesil-miR-21。使用脂质体Lipofectamine2000转染小鼠肾小球系膜细胞后,G418筛选,获得稳定转染克隆,提取总RNA,通过实时荧光定量RT-PCR技术检测miR-21的表达。结果经酶切鉴定和测序证实,合成的miR-21基因序列完全正确,并已成功克隆到真核表达质粒pGenesil-1上。重组真核表达质粒转染小鼠肾小球系膜细胞后,筛选出的阳性克隆可稳定高表达miR-21。结论已成功构建miR-21真核表达质粒,并在小鼠肾小球系膜细胞中高效表达,为进一步探讨miR-21的生物学功能奠定了基础。

Construction of Eukaryotic Expression Vector for Mouse MicroRNA miR-21 and Its Expression in Mouse Mesangial Cells

Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and express in mouse mesangial cells.Methods The miR-21 gene was chemically synthesized and cloned into expression vector pGenesil-1.The constructed recombinant plasmid pGenesil-miR-21 was transfected to mouse mesangial cells in mediation of Lipofectamine 2000,and the stably transfected clones were screened with G418,from which total RNA was extracted for determination of expression of miR-21 by real-time fluorescent quantitative RT-PCR technique.Results Both restriction analysis and sequencing proved that recombinant plasmid pGenesil-miR-21 was constructed correctly.The miR-21 gene was stably and highly expressed in the screened positive clones of mouse mesangial cells transfected with the constructed recombinant plasmid.Conclusion The eukaryotic expression vector for miR-21 was successfully constructed and highly expressed in mouse mesangial cells,which laid a foundation of further investigation of biological function of miR-21.