Transgenic line construction and initial genetic analysis. (A) Expression construct used with the murine GPIbα proximal promoter shown in black and the hB^-FVIII in white. The SV40 5′-UTR and 3′-UTR are shown in gray. Primer pairs for the genomic analysis and for RT-PCR analysis are shown below. X indicates XhoI; S, SalI. (B) Genomic Southern blot after Bgl II digestion and probed for the murine GPIbα promoter region is shown on the left and the schematic representation of the probe and expected bands on the right. Calculated copy number for the transgene per haploid genome determine by phosphorimaging analysis is shown below the Southern blot. The position of the Bgl II site immediately upstream of the GPIbα transcriptional start site, and the ends of the probe are noted. B indicates Bgl II. (C) Platelet RT-PCR from various founder lines for the transgene is positive for multiple lines as shown in the top gel. The expected 0.3-kb cDNA band is indicated. In some lanes, a 0.6-kb genomic band is seen. The bottom gel is an RT-PCR amplification of PF4 message, confirming the platelet nature of the RNA. M indicates ϕX HaeIII marker; WT, wild-type mice platelet RNA; and H₂O, lane with no RNA added. (D) In the top gel, RT-PCR on total RNA extracts from various tissues were studied for line no. 38, demonstrating that the only tissue that was positive for recombinant hB^-FVIII message amplification of the transgene was the bone marrow. At the bottom, virtually all the tissues tested were positive for the widely expressed message of murine HPRT. M indicates ϕX HaeIII marker; br, brain; t, tongue; lu, lungs; h, heart; li, liver; sm, stomach; sp, spleen; si, small intestines; kd, kidney; te, testicle; sk, skeletal muscle; and bm, bone marrow.