[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.
Ma
Qiang
Q
College of Biotechnology, Southern Medical University, Guangzhou 510515, China.
Li
Ming
M
Dong
Wen-Qi
WQ
Wu
Ying-Song
YS
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