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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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【References】
Chinese Journal Full-text Database 3 Hits
1 YU Xiao-Lin, LIU Hong, CHEN Wan-Ping, CHENG Peng-YuanOutpatient Department, Fourth Military Medical University, Xi'an 710033, China;Effects of recombinant Caspase-3 on human laryngocarcinoma cell line Hep-2 through coexpression with GFP[J];Journal of The Fourth Military Medical University;2005-21
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3 WANG xiao-xia1,2,YAO xiao-bao1, JI xian-sheng2, ZHU hong-liang1 1Department of Otolaryngology, First Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710061, China;2Department of Otolaryngology, 451 Hospital of PLA, Xi'an 710054, China;Effect of recombinant caspase-3 gene transfection on CNE2 cell apoptosis[J];Journal of Southern Medical University;2009-01
【Co-references】
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1 CHEN Jian jing1*, Raab Traub Nancy2, YAO Qing yun3, ZHANG Feng1,HUANG Mei ling1, KUANG Zhu ji1, ZHANG Xiao shi1, YE Yan li1, GU Li11.Cancer Center, Zhongshan University,Guangzhou 510060, P.R.China2.Cancer Center, North Carolina University, Chapel;Inhibiting Target Gene Expression and Controlling Growth of Epstein-Barr Virus Transformed Cells by Antisense RNA Transcripts[J];Chinese Journal of Cancer;2002-01
2 JIA Lin Tao, YU Cui Juan, XU Yan Ming, ZHU Feng, PENG Wei Dan, SU Cheng Zhi, WANG Cheng Ji, YANG An Gang Department of Biochemistry and Molecular Biology, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi'an 710033, China;Construction and effect of caspase-3 fusion proteins and their apoptosis induction in HeLa cells[J];Journal of The Fourth Military Medical University;2001-12
3 ZHANG Yong 1, JIA Lin-Tao 1, LI Qing-Xia 1, WANG Cheng-Ji 1, ZHOU Shi-Sheng 3, YANG An-Gang 1,2 1Department of Biochemistry and Molecular Biology, 2Department of Immunology, School of Basic Medicine, Fourth Military Medical University, Xi'an 710033, China, 3Institute of Basic Medical Sciences, Medical Collage, Dalian University, Dalian 116622, China;Vector construction and silencing effect of volume-regulated chloride channel gene-targeted small interfering RNA[J];Journal of The Fourth Military Medical University;2005-09
4 TANG Ni *, HUANG Ai-long, ZHANG Bing-qiang, YAN Ge, Tong-Chuan He. *Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China Corresponding author: HUANG Ai-long 400010, Institute for Viral Hepatitis, Chongqing University of Medical Sciences;Potent and specific inhibition of hepatitis B virus antigen expression by RNA interference[J];National Medical Journal of China;2003-15
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1 ZHAO Jun-Xia, ZHENG Li-Fen, ZHAO Juan, SHEN Yuan, WANG Yan-Ling, YAN Yun-Li Department of Cell Biology, Basic Medical College, Hebei Medical University, Shijiazhuang, 050017, China;Effects of pholiota nameko polysaccharides on K562 leukemia cell proliferation and Caspase-3 expression in vitro[J];Journal of the Fourth Military Medical University;2007-15
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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