RT-PCR analysis of hPBP versus mPBP.

(A) Ethidium bromide analysis of platelet RT-PCR products for mPBP (top) and for hPBP (bottom) for all of the founder lines studied plus wild-type (WT) mouse platelets and the appropriate cDNA control are included. The founder lines are numbered by transgenic copy. The PCR amplification was carried out for 30 cycles. The anticipated cDNA band is indicated by a large arrow. The genomic DNA band is indicated by a smaller arrow and is only occasionally prominent when there is no or little RNA message. No cDNA bands were seen when the RT step was skipped or when RNase A was included in the RT step (data not shown). WT indicates wild-type litter mates; M, φX HaeIII marker. (B) Relationship between the copy number for the PBP-expression transgenic lines Short-PBP, Long-PBP, andλ-PBP/PF4 and the level of hPBP versus mPBP message are shown. The relative quantity of hPBP to mPBP message was determined using Cy5-labeled primers in the amplification step and measuring final product quantitatively with a Storm imager. Analysis was done of the PCR products within the logarithmically amplifying range of cycles (usually 14-20 cycles) corrected for the relative efficiency of the 2 sets of primer pairs; ▪, Short-PBP; ●,Long-PBP; ♦, λ-PBP/PF4. Data are shown for average ± 1 SD. Experiments were repeated 3 separate times.