[Study on high-efficiency gene transfer of pseudotyped HIV vector].
OBJECTIVE
To investigate the high-efficiency of pseudotyped HIV as gene transfer vector.


METHODS
Three plasmids of pseudotyped HIV gene transfer vector system were transferred into packaging cell line 293T by Ca3 (PO4)2 precipitation method. GFP (Green Fluorescence Protein) or HSV-tk gene was constructed in the plasmid pHR'CS respectively (pHR'CS.GFP or pHR'CS.HSV-tk). The pseudotyped HIV particles were observed through electronic microscopy and were measured through spectrofluorophotometer. High titer pseudotyped HIV was harvested from volume of virus-producing cell supernatant and concentrated. Ovarian epithelial cancer cell line SKOV3 and normal human gingival fibroblast cell GF were infected by pseudotyped HIV. PCR and RT-PCR were resorted to demonstrate the successful transduction and transcription of the HSV-tk gene. After administration of GCV, the changes of those cells and apoptosis were observed through optical microscopy. The cytotoxicity efficacy of HSV-tk/GCV system was evaluated by MTT method. The growth inhibition rate (GIR) of cells and inhibition concentration 50 (IC50) were counted.


RESULTS
The above plasmids were effectively transferred into 293T cell. A lot of pseudotyped HIV particles were observed through electronic microscopy. The virus supernatant had a high absorbing value at 510 nm through spectrofluorophotometer, which proved the existence of virus. After pseudotyped HIV infection, SKOV3 and GF had remarkable infection rate. 600 bp strand was seen through PCR and RT-PCR. Changes and apoptosis of cells followed by administration of GCV were observed. The MTT method showed that the cytotoxicity efficacy of GCV was high to SKOV3 and GF cell.


CONCLUSIONS
The pseudotyped HIV is a high-efficiency gene transfer vector.
Liu
C S
CS
Bioengineering Center, Qilu Hospital of Shandong University, Jinan 250012, China. chunsheng_l@yahoo.com
Song
L
L
Kong
B H
BH
Ma
D X
DX
Wei
M
M
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D041722:Genes, Transgenic, Suicide / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006678:HIV / Q000235:genetics; D006801:Humans; D014162:Transfection