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《Journal of Peking University(Health Sciences)》 2005-02
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Helper-virus-free herpes simplex virus-1 vector mediated LacZ gene expression in cultured cortical neurons

WANG Han-sen, PAN Hong, ZHANG Yu-zhi, BAO Xin-hua, ZHANG Guo-rong, WU Xi-ru △(Department of Pediatrics, Peking University First Hospital, Beijing 100034, China)  
Objective: To obtain knowledge about the helper-virus-free packaging system of HSV-1 vector and its use in cultured neurons by investigating helper-virus-free packaging of HSV-1 vector and LacZ gene expression mediated by the vector in rat cultured cortical neurons. Methods: The cosmids with inserts of HSV-1 genome were digested by Pac Ⅰ, purified and cotransfected with HSV plasmid vector DNA (with LacZ gene) into 2-2 cells. After 3 hour incubation at 37 ℃, the media were changed into DMEM with 6%CCS. And the 2-2 cells were incubated for 72 hours at 34 ℃. Then the virus particles were harvested. The medium with the virus particles was added to BHK cells. After 24 hour further culture, X-gal staining was performed. The virus particles were added to 3 day cultured cortical neurons. After 1, 7 and 14 day further cultures, neurons were stained by X-gal and observed under microscope. Results: The BHK cells (stained blue) expressing LacZ gene could be seen after 24 hour further culture. After 1, 7 and 14 day further cultures, neurons stained blue by X-gal staining could be seen all the time in different groups. Conclusion: Helper-virus-free package system can produce effective virus particles from HSV-1 plasmid vector with A tyrosine TH-NF chimeric promoter and mediate stable exogenous gene expression in cultured neurons, thus providing a useful tool for gene transfer and study of gene function in the nervous system.
【Fund】: 国家自然科学基金(30200152;; 30271374 );; 国家“211”工程北京大学“十五”重点学科建设项目资助~~
【CateGory Index】: R346
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