Fig. 1. 2bF8 transgene analysis.
(A) Diagram of the pWPT-2bF8/MGMT construct. FVIII is driven by the platelet-specific αIIb promoter. MGMTp140k was driven by the MSCV promoter. (B) PCR detection of the 2bF8 transgene. DNA was purified from peripheral white blood cells. A 0.65 kb fragment from the 2bF8 expression cassette was amplified. Wild type (WT) mFVIII and mFVIIInull PCRs were used as controls to confirm that 2bF8/MGMT-transduced recipients were on the FVIIInull background. (C) qPCR determination of the average copy number of the 2bF8/MGMT transgene per cell in transduced recipients. Peripheral blood cell-derived DNA was analyzed for the 2bF8 transgene and normalized to the ApoB gene. These results demonstrate that 2bF8/MGMT genetically modified hematopoietic cells were viable and selectable by BG/BCNU treatments.