Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation

• Published in: Journal of virological methods 1997, Vol.63 (1), p.129-136
• Main Authors: Maxwell, Françoise, Harrison, Gail S., Maxwell, Ian H.
• Format: Article
• Language: English
• Subjects: Adeno-associated virus; Alkaline phosphatase; Biological and medical sciences; Biotechnology; Cell Line; Cell Line, Transformed; Dependovirus - genetics; Electroporation; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Genetic engineering; Genetic technics; Humans; Infectious titer; Luciferase; Luciferases - genetics; Methods. Procedures. Technologies; Microbiology; Recombination, Genetic; Techniques used in virology; Transfection; Tumor Cells, Cultured; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(96)02121-0

Adeno-associated virus (AAV) is useful as an integrating vector for gene transfer. AAV recombinants are generally produced by transient co-transfection methods since it has proven difficult to generate stable packaging cell lines. Acceptable titers of transducing recombinants should be obtainable by optimizing conditions for transient co-transfection. Here, using a luciferase reporter derivative of the AAV infectious plasmid p sub201, we show that substantially higher yields of transducing virus can be obtained using electroporation, rather than calcium phosphate transfection. Furthermore, we observed that electroporation of NB324K cells (an SV40-transformed human cell line) with the helper plasmid, pAAV/Ad, with concomitant adenovirus dl309 infection, gave yields of luciferase transducing recombinant AAV equal or superior to those obtained from the more commonly employed 293 cells. NB324K cells are easier to manipulate and show increased cell-association of the recombinant virus (facilitating its concentration and purification). We also adapted an in situ infected cell hybridization procedure, using a digoxigenin labeled probe, as a general method for determining infectious titer. Titers thus estimated were similar for luciferase-transducing and for alkaline phosphatase-transducing AAV vectors; the estimated titer of the latter agreed with that determined by in situ expression of alkaline phosphatase. We also describe a multiple cloning site derivative of p sub201 which should facilitate generation of further AAV recombinants.