Generation of 2bF9 transgenic mice. (A) Schematic representation of 2bF9 lentiviral vector. A 2.8-kb hFIX cDNA, including a 1.4-kb truncated intron 1 between exon 1 (E1) and the remaining exons (E2-8) is under control of the αIIb promoter (αIIb^Pr). (B) PCR analysis of mouse genomic DNA showing that 2bF9 transgene is detected in 5 founder mice. The expected 0.35-kb band is seen. Lanes 1 to 5 indicate 5 founder mice; lane 6, FIX^null mouse control; lane 7, pWPT-2bF9 plasmid DNA control; lane 8, water control; and M, Hi-Lo DNA marker. (C) FIX expression in the platelets of 5 founders. Founders HW1473 and HW1474 were developed into the 2 transgenic lines referred to in Figure 2A as 2bF9-H and 2bF9-L, respectively. plts indicates platelets. (D) Integration sites of 2bF9 transgene. (Top) 2bF9 transgene integration site of 2bF9-L mice in the third intron of the Samd4b gene on chromosome 7. Transgene is in reverse transcriptional orientation to Samd4b. (Bottom) Transgene integration site of 2bF9-H mice between the hypothetical gene D15Mgi27 and the Espl1 gene on chromosome 15. 2bF9 was oriented reverse to both genes.