[A novel system for producing lentiviral vectors].
The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamine2000. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT-PCR and the Western blot, the results showed that lentiviral vectors were found in the culture supernatant; approximately (11.71 +/- 0.80) x 10(11) copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real-time PCR; the vector stocks with titers was up to (1.3 +/- 0.18) x 10(8) tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.
College of Biotechnology, Southern Medical University, Guangzhou 510515, China.
Qiang
Q
Ma
Ming
M
Li
Wen-Qi
WQ
Dong
Ying-Song
YS
Wu
D018929:Cell Culture Techniques / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005822:Genetic Vectors; D006378:Helper Viruses; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases