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《Journal of Nanchang University(Medical Science)》 2012-08
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Analysis and Selection of Human Nuclear Distribution Protein C-specific shRNA by Flow Cytometry

LIAO Xia,ZOU Yu-guang,PANG Shi-feng,ZHENG Ke-qin,ZHOU Ru-bin, CAO Ding-guo,LIANG Peng,HU Chuan-yin(Department of Biology,the Basic Medical College of Guangdong Medical College, Zhanjiang 524023,China)  
Objective To select the effective human nuclear distribution protein C(hNUDC)-specific shRNA using flow cytometry through constructing vectors pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Methods NUDC-RFP fused gene was cloned into pDs vector,resulting in pDs-NUDC-RFP.Human U6 promoter and hNUDC-specific shRNA were cloned into pDs-NUDC-RFP vector,resulting in pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Then 293T cells were transfected with high-quality plasmid by liposome complex method.The fluorescence intensity and the number of fluorescent cells were detected by flow cytometry.NUDC-RFP mRNA was analyzed by quantitative real-time PCR and NUDC-RFP protein was detected by Western Blot.Results The 293T cells in blank control group did not show RFP fluorescence.However,a large number of cells in negative control group showed strong expression of RFP,but the fluorescence intensity and the number of fluorescent cells were significantly reduced in 293T cells transfected with hNUDC-specific shRNAs compared with negative control group.The levels of NUDC-RFP fusion protein expression were decreased by 90% and 85% in cells transfected with shNUDC-F and cells transfected with shNUDC-A,respectively.In addition,NUDC-RFP expression was down-regulated by 62%-75% by transfection with pDs-NUDC-RFP-TK-U6-shNUDC-B,C,D,E and H,respectively.NUDC-RFP gene expression was not disrupted in negative control group.There results were consistent with real-time RT-PCR and Western Blot analysis and showed that shNUDC-F was the best effective interference fragment.Conclusion Flow cytometry analysis is an effective method for selecting shRNA vector with fluorescent marker.
【Fund】: 教育部科学技术研究重点项目(210156);; 广东省卫生厅青年基金(B2010235);; 广东医学院博士启动基金(XB1007);; 广东省大学生创新实验项目(KY1003);; 湛江市科技攻关项目(2010C3111005)
【CateGory Index】: R346
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【Citations】
Chinese Journal Full-text Database 4 Hits
1 YANG Xu-hui1,2,XIA Tian2,5,YU Wei-hua2,ZHONG Yue-si3,XIANG Peng2,HE Feng4(1.Assisted Reproductive Center,Guangdong Women and Children Hospital,Guangzhou 510010,China;2.Center for Stem Cell and Tissue Engineering,Sun Yat-sen University,Guangzhou 510080,China;3.Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China;4.School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou 510006,China;5.Taizhou Hospital of Zhejiang Province,Linhai 317000,China);Construction of human nestin specific short hairpin RNA expressing vector and screening of stable transfected cell clone[J];Journal of Guangdong Pharmaceutical University;2012-02
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【Co-citations】
Chinese Journal Full-text Database 3 Hits
1 ZOU Yuguang1,MO Yuye1,PANG Shifeng1,ZHANG Qiang2,YANG Fang3, YI Wenjun1,HE Guofeng1,YU Weiwei1,ZHENG Keqin1(1.Department of Biology,Guangdong Medical College,Zhanjiang 524023,Guangdong,China;2.Mai Rui Biology Medical Treatment Electron Limited Company of Shenzhen,Shenzhen 518057,Guangdong,China; 3.Yushan Middle School of Guangzhou City,Guangzhou 511400,Guangdong,China);Construction of Mpl Gene RNA Interference Vector and Selecting Effective Mpl-specific shRNA with Fluorescence Microscopy[J];Journal of Guiyang Medical College;2012-06
2 LIAO Xia1,PANG Shi-feng1,ZHANG Qiang2,YANG Fang3,ZHENG Ke-qin1,ZHOU Ru-bin1,CAO Ding-guo1,ZOU Yu-guang11Department of Biology of Guangdong Medical College,Zhanjiang 524023;2Mai Rui Biology Mdical Treatment Electron Limited Company of Shenzhen,Shenzhen 518057;3Yushan Middle School of Guangzhou,Guangzhou 511400,China;The construction of NUDC shRNA interference vector with red fluorescence protein[J];Chinese Journal of Cellular and Molecular Immunology;2011-07
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