A novel system for producing lentiviral vectors.
- Author:
Qiang MA
1
;
Ming LI
;
Wen-Qi DONG
;
Ying-Song WU
Author Information
1. College of Biotechnology, Southern Medical University, Guangzhou 510515, China.
- Publication Type:Journal Article
- MeSH:
Cell Culture Techniques;
methods;
DNA-Directed RNA Polymerases;
genetics;
Genetic Vectors;
Helper Viruses;
Lentivirus;
genetics;
growth & development;
Plasmids;
genetics;
Recombination, Genetic;
Reverse Transcriptase Polymerase Chain Reaction;
methods;
Transfection;
Vaccinia virus;
genetics;
Viral Proteins;
genetics
- From:
Chinese Journal of Biotechnology
2007;23(5):958-960
- CountryChina
- Language:Chinese
-
Abstract:
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.
