SB mediates long-lasting hFVIII expression in vitro. 1 × 105 HeLa cells were seeded in 6-cm dishes and transfected with 1 μg pSBT/mCAGF8 with either 1 μg of pUB-SB10 (▦) or pGFP(▴; as a control for random integration). One milliliter of media was collected before transfection, 24 hours after transfection, and every week thereafter; hFVIII levels were measured by ELISA. The transfected cells were subcultured 1:10 starting 1 week after transfection and every week thereafter. Without SB transposase, hFVIII levels in the media were undetectable 2 weeks after transfection, thus showing an SB-dependent positive effect on hFVIII gene expression. (n = 3/group; error + SD).