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《Journal of The Fourth Military Medical University》 2005-08
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Construction and identification of RNA interference expression vector targeting EGFP

MA Long-Yang 1,LIU Jia-Yun 2,XU Yan-Ming 1,JIA Lin-Tao 1,WANG Cheng-Ji 1,YANG An-Gang 1,2 1Department of Biochemistry & Molecular Biology, 2Department of Immunology,School of Basic Medicine,Fourth Military Medical University,Xi’an 710033,China  
AIM: To construct pSUPER RNA interference expression vector targeting enhanced green fluorescent protein(EGFP) and to test the silencing effect in mammalian COS-7 cell line.METHODS: The designed oligos targeting EGFP were cloned into the pSUPER vector,which was lineated after BglⅡ and HindⅢ digestion.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombinant plasmids and pIRES2-EGFP were co-transfected into COS-7 cells using Lipofectamine 2000 .The suppression effect of EGFP was assayed by fluorescence microscope 48 h after transfection. RESULTS: The pSUPER siRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and the DNA sequencing.The over-expression of EGFP was detected in the COS-7 cells which were transfected pIRES2-EGFP or co-transfected pIRES2-EGFP and pSUPER.In comparison with that of the control group,the expression of EGFP in COS-7 cells which were co-transfected pIRES2-EGFP and pSUPER-R237 or pSUPER-R304 or pSUPER-R447 was weaker and the number of cells emitting green fluorescence decreased distinctly.CONCLUSION: pSUPER siRNA expression vectors targeting EGFP are successfully constructed.The expression of EGFP gene is inhibited effectively in mammalian cells which are co- transfected with pIRES2-EGFP and pSUPER-Rs.
【Fund】: 国家 973基金 (2004CB518805);; 国家 863基金(2004AA217 071);; 国家自然科学基金(30200274)
【CateGory Index】: Q78
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2 YAO Yu,HAI Ya-Nan,NAN Ke-Jun,YUAN Zhang-Li,NIE Yan-Li Department of Oncological Medicine,First Affiliated Hospital,Medical College,Xi'an Jiaotong University,Xi'an 710061,China;Effect of Delisheng on apoptosis of liver cancer HepG2 cell line and its mechanism[J];Journal of the Fourth Military Medical University;2009-18
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