title: Optimized production and concentration of lentiviral vectors containing large inserts.
abstract: Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.
authors.affiliation: Department of Dermatology, Charité University Hospital, Berlin, Germany.
authors.forename: Nadya
authors.initials: N
authors.lastname: al Yacoub
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Romanowska
authors.forename: Natalie
authors.initials: N
authors.lastname: Haritonova
authors.forename: John
authors.initials: J
authors.lastname: Foerster
mesh_terms: D004247:DNA; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development*; D014162:Transfection; D014461:Ultracentrifugation
chemical_list: D004247:DNA