Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer
Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vect...
| Publié dans: | BioTechniques. - 1993. - 34(2003), 5 vom: 14. Mai, Seite 1074-8, 1080 |
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| Auteur principal: | |
| Autres auteurs: | , , |
| Format: | Article |
| Langue: | English |
| Publié: |
2003
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| Accès à la collection: | BioTechniques |
| Sujets: | Evaluation Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. |
| Résumé: | Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells |
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| Description: | Date Completed 20.02.2004 Date Revised 10.12.2019 published: Print Citation Status MEDLINE |
| ISSN: | 0736-6205 |