首页|期刊导航|中国神经再生研究（英文版）|Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation
中国神经再生研究（英文版）2008，Vol.3Issue(4)：456-460,5.
Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation
Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation
摘要
Abstract
BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION: We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.关键词
glucose transporter-1/ cloning/ recombinant adenoviral vectorKey words
glucose transporter-1/ cloning/ recombinant adenoviral vector分类
医药卫生引用本文复制引用
Fangcheng Li,Junliang Li,Ranyi Liu,Xinke Xu,Kaichang Yuan,Zhonghua Wu..Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation[J].中国神经再生研究（英文版）,2008,3(4):456-460,5.基金项目
the National Natural Science Foundation of China, No. 39900048 （）
the Natural Science Foundation of Guangdong Province, No. 010721 （）
