efVIII variant is expressed more efficiently than sfVIIIΔB in 293T and K562 cells. (A) efVIII and sfVIIIΔB expression levels in transiently transfected 293T cells. 293T cells were transiently transfected using 10 μg MSGV-sfVIIIΔB (column 1), FB.sfVIIIΔB (column 2), or FB.efVIII (column 3) plasmid DNAs. At 36 hours later, the conditioned medium was assayed for fVIII antigen levels. *Significantly higher levels (P < .001). (B) efVIII and sfVIIIΔB expression levels in stably transduced K562 cells. K562 cells were transduced with the FB.efVIII or MSGV-sfVIIIΔB vectors at an MOI of 1. DNA was isolated from the transduced cells 2 weeks later, digested with ApaI and BstBI, and subjected to Southern blot analysis. Pools of stably transduced cells with an average of one vector copy per cell were selected and their conditioned medium assayed for fVIII antigen levels. The standards were generated by digesting MSGV-sfVIIIΔB plasmid DNA in amounts equivalent to 0.1, 1, and 10 vector copies per cell. Vector copy numbers were calculated by PhosphoImager quantification and comparison with copy number standards and normalization to GAPDH controls. The approximate vector copy numbers per cell are indicated below the lanes. fVIII antigen levels were determined by a commercial ELISA (fVIII:C Ag). Data presented are the mean values of several independent experiments (n = 3) ± SDs. *Significantly higher levels (P < .001).