2bF8 transgene analysis. (A) PCR detection of 2bF8 transgene. DNA was purified from peripheral white blood cells. A common antisense primer and a sense transgene-specific primer were used to amplify a 0.292-kb fragment from the 2bF8 transgene. The same common antisense primer and a wild-type allele-specific sense primer were used to amplify a 0.207-kb fragment from the wild-type allele. (Left panel) Results from recipients before BM transplantation. (Right panel) Results from recipients at least 3 weeks after BM transplantation. Shown is one representative experiment that was performed 3 times. (B) Real-time PCR determined the average copy number of the 2bF8 transgene per cell in BMT recipients. DNA was purified from peripheral white blood cells, and 100 ng of DNA was analyzed for the 2bF8 transgene, with normalization to the Apo B. Bars represent mean plus or minus SD. Data shown are summarized from 10 BM transplantation trials. These results demonstrate viable engraftment of 2bF8 genetically modified HSC in immunized recipients.