Construction and identification of RNA interference expression vector targeting EGFP

    MA Longª²Yang1,LIU Jiaª²Yun2,XU Yanª²Ming1,JIA Linª²Tao1,WANG Chengª²Ji1,YANG Anª²Gang1,2

    1Department of Biochemistry £¦ Molecular Biology,2Department of Immunology,School of Basic Medicine,Fourth Military Medical University,Xi¡¯an 710033,China

    ¡¾Abstract¡¿AIM£º To construct pSUPER RNA interference expression vector targeting enhanced green fluorescent protein(EGFP) and to test the silencing effect in mammalian COSª²7 cell line.METHODS£º The designed oligos targeting EGFP were cloned into the pSUPER vector,which was lineated after Bgl¢ò and Hind¢ó digestion.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombinant plasmids and pIRES2ª²EGFP were coª²transfected into COSª²7 cells using Lipofectamine2000.The suppression effect of EGFP was assayed by fluorescence microscope 48 h after transfection.RESULTS£º The pSUPER siRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and the DNA sequencing.The overª²expression of EGFP was detected in the COSª²7 cells which were transfected pIRES2ª²EGFP or coª²transfected pIRES2ª²EGFP and pSUPER.In comparison with that of the control group,the expression of EGFP in COSª²7 cells which were coª²transfected pIRES2ª²EGFP and pSUPERª²R237 or pSUPERª²R304 or pSUPERª²R447 was weaker and the number of cells emitting green fluorescence decreased distinctly.CONCLUSION£º pSUPER siRNA expression vectors targeting EGFP are successfully constructed.The expression of EGFP gene is inhibited effectively in mammalian cells which are coª² transfected with pIRES2ª²EGFP and pSUPERª²Rs. ......