Construction of high-titer clinical vector producer cells. (A) Distribution of calculated ED7R cell titer of supernatants from 20 independent producer clones for SCID-X1 clinical vector. Each point represents an individual clone, and the horizontal bar shows the average of the titers from all of the clones. The dashed line indicates the highest titer achieved for an identical vector prepared using 4-plasmid transient transfection of 293T cells. (B) Southern blot analysis of ED7R genomic DNA after transduction with 50 μL supernatant from the top 4 cloned producer cell lines (lanes 1-4, in order of decreasing titer) and from transiently transfected 293T cells (lane T). The SbfI enzyme used cuts once in each copy of the chromatin insulator, and it is expected to generate a 4-kb fragment spanning the complete provirus. (C) Flow cytometry histograms of human common γc antibody binding to ED7R cells 4 days after incubation with either fresh media (Mock), or 5 μL supernatant produced by the top performing stable producer cell clone.
