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《Chinese Journal of Neuromedicine》 2007-11
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Optimal screening of shRNA molecules and reconstruction of the shRNA eukaryotic expression vector of GFAP expressive suppression of rat spinal cord-derived astrocytes

LI Zhen-yu GAO Ming-yong XIAO Jian-de YAN Hong-yin.Department of Spine Surgery,the Affiliated Shenzhen Hospital of Southern Medical University,Shenzhen 518035,China  
Objective To design and screen short hairpin RNA(shRNA)molecules targeting the coding region of the rat glial fibrillary acidic protein(GFAP)gene,and reconstruct eukaryotic expression vector of shRNA.Methods Three pairs of shRNA templates which composed of 19 base pair(bp)reverse repeated motifs of GFAP target sequence with 9 bp spacer were designed and synthesized,and then inserted directionaUy into plasmid pSilencer2.1 respectively to generate small interfering RNA(siRNA)eukaryotic expression vector.The shRNA molecules were treated with liposome-mediated transfection via the ex vivo expression repressive model of GFAP of rat spinal astrocytes;the effects of GFAP expressive suppression of primarily-cultured astrocytes were detected with real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blot,and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.Results Sequence analysis showed that the recombinant eukaryotic expression vector of GFAP-shRNA was successfully constructed.The optimal GFAP-shRNA eukaryotic expression vector was screened using real-time fluorescent quantitative RT-PCR and Western blot.The expressions of GFAP were reduced by 81%,63% and 56% respectively at the expressive level ofmRNA and protein. Conclusion The GFAP-gene expression can be effectively suppressed by the GFAP-shRNA eukaryotic expression recombinant optimized via the ex vivo cellular expression repressive model of GFAP,paving the way for the application of the following multiple targets of RNA interference(RNAi)genetic manipulation in the treatment of suppression of glial scar formation after spinal cord injury.
【CateGory Index】: Q78
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Chinese Journal Full-text Database 2 Hits
1 ZHANG Yan1, XUE Yao-ming1, GUO Ai-lin2, ZHANG Wen-min2 1Department of Endocrinology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2Department of Pathology, Sen Yet-sen University, Guangzhou 510089, China;Prokaryotic expression vector construction, expression and polyclonal antibody preparation of the fusion protein of glutathione S-transferase and peroxisome proliferator-activated receptor-gamma coactivator-1[J];Journal of First Military Medical University;2005-05
2 HE Xiang-liang1,HE Dong-hua2,LI Feng3 ,YANG Chuan3,CHENG Hua3,FU Zu-zhi 3 (1Department of Emergency,The First Affiliated Hospital,2.Da An Gene Co mpany,3.Department of Endocrinology,The Second Affiliated Hospital,SUN Yat-sen University,Guangzhou 510080,China);Construction and Expression of A Prokaryotic Expression Vector Containing Human Resistin Gene[J];Journal of Sun Yat-sen University (Medical Sciences);2004-05
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1 TONG Tie-gang1, LIN Yan1,2, MU Dan-mei3,4, BAI Yu1, YANG Mu-lei3, ZHENG Min3, WU Dong-lai1 1National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2College of Life Sciences, Heilongjiang University, Harbin 150080, China; 3First Clinical Medical College of Harbin Medical University, Harbin 150001, China; 4Department of Abdominal Ultrasonagraphy, Daqing Oilfield General Hospital, Daqing 163001, China;Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2[J];Journal of Southern Medical University;2009-06
2 WANG Zhihui 1,2) , WU Yiming 1) ,WU Yongjun 1) 1)Department of Occupational Health, College of Public Health, Zhengzhou University, Zhengzhou 450052 2)Department of Occupational Health,Henan Institution of Professional Medicine, Zhengzhou 450052;Cloning, expression and purification of human glutathione S-transferase Pi[J];Journal of Zhengzhou University(Medical Sciences);2006-06
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