Conditional F8-KO alleles. (A) Targeted F8 gene. (B) F8^F allele. Excision of the neomycin resistance cassette by Flp recombinase produces the floxed (F8^F) allele, which is expressed normally. (C) F8^KO allele. Excision of exons 17/18 from the F8^F allele by Cre recombinase produces a F8^KO allele. (D) Alternative mRNA splicing of the F8^KO allele. The predicted exon 16/19 splice [F8^KO(16/19)] and an alternatively spliced transcript [F8^KO(Alt)] in which 46 bp of intron 16 is retained are produced in approximately equal amounts. (E) Domain structure of normal FVIII protein. The position of exons 17/18 within the A3 domain is noted. (F) Predicted FVIII polypeptide encoded by F8^KO(16/19) mRNA. (G) Predicted FVIII polypeptide encoded by F8^KO(Alt) mRNA. Flp recombinase target (FRT) sites are represented as open triangles, and Cre recombinase target (loxP) sites as gray triangles. The positions of duplex genotyping primers P1, P2, and P3 and RT-PCR primers P4 and P5 are shown. aa, amino acid; Ex, exon.