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.