Heterologous expression of fVIII transgenes. (a) Schematic of fVIII subunits. Full-length human with activation peptide indicated (top), B-domain deleted (BDD) human with the SQ-linker indicated (middle-top), BDD-porcine (middle-bottom), and HP-fVIII (bottom). Dark gray denotes porcine-specific sequences. (b) Human (BDDhfVIII), porcine (BDDpfVIII), and HP-fVIII transgenes were transfected into both BHK-M and Flp-In 293 cells. BHK-M cells were modified using Lipofectamine 2000 and ReNeo plasmid constructs. Flp-In 293 cells were modified by FLP recombinase-mediated site-specific recombination then screened by Southern blot in order to compare only those clones containing a correctly integrated single transgene. FVIII activity was measured from 19 to 33 BHK-M clones (black circles) and 13–22 singly integrated Flp-In clones (white circles) by one-stage coagulation assay. Mean HP-fVIII activity is depicted by a horizontal dotted line.