[Helper-virus-free herpes simplex virus-1 vector mediated LacZ gene expression in cultured cortical neurons].
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 degrees C, the media were changed into DMEM with 6% CCS. And the 2-2 cells were incubated for 72 hours at 34 degrees C. 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.


CONCLUSIONS
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.
Wang
Han-sen
HS
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Pan
Hong
H
Zhang
Yu-zhi
YZ
Bao
Xin-hua
XH
Zhang
Guo-rong
GR
Wu
Xi-ru
XR
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006378:Helper Viruses; D018259:Herpesvirus 1, Human / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar