A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors
Zhang, B., Xia, H.Q., Cleghorn, G. J., Gobe, G., West, M., & Wei, M.Q. (2001) A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors. Gene Therapy, 8(22), pp. 1745-1751.
Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.
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|Item Type:||Journal Article|
|Keywords:||Biochemistry & Molecular Biology, Biotechnology & Applied Microbiology, Genetics & Heredity, Medicine, Research & Experimental, Lentiviral Vector, Concentration, Poly-l-lysine, Large Volume, High Titre, Cationic Liposomes Enhance, Murine Leukemia-virus, Gene-transfer, In-vivo, Stable Transduction, Nondividing Cells, High-titer, Retrovirus, Vitro, Hiv|
|Divisions:||Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Current > Schools > School of Exercise & Nutrition Sciences
|Copyright Owner:||Copyright 2001 Nature Publishing Group|
|Deposited On:||28 Oct 2015 00:10|
|Last Modified:||28 Oct 2015 00:16|
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