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Figure 1 | BMC Proceedings

Figure 1

From: Novel approaches to render stable producer cell lines viable for the commercial manufacturing of rAAV-based gene therapy vectors

Figure 1

(A) Transfection- and infection-based generation of rAAV in HeLa cells at different temperatures. Cells were transfected by calcium phosphate using three plasmids encoding the rAAV vector, rep and cap, followed by AdV5 infection and subsequent incubation of the cells at three different temperatures. Genomic rAAV titers were determined 96 h post infection as previously described [4]. (B) Investigation of rAAV production in a "transfection only approach"applying plasmids encoding rep, cap, vector as well as adenoviral E1 and remaining AdV helper functions. Different variants of rep and cap were compared regarding rAAV productivity. 1: Approach implying functionally separated rep and cap genes on different plasmids, which are devoid of rep78 expression and lack an artificial Rep Binding Site (RBS) in the pUC19 plasmid backbones [6] (standard plasmids used in all preceding experiments). 2: Same rep and cap plasmids but modified to avoid the expression of non-functional and truncated viral gene products by deletion of various promoter and potential transcription start sites. Genome titre was analyzed 120 h post infection as previously described [4].

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