Kinetics of appearance and disappearance of marked cells in recipients of retrovirally transduced mobilized PB cell autografts. (A) Time course studies revealing an early contribution of marked cells to the WBCs produced in the first 3 weeks in all 3 patients studied with variable patterns of contribution at later times. DNA was extracted from sequentially obtained samples of PB cells from each patient, and proviral sequences were detected using primers designed to amplify a 589-bp 5′ vector sequence. Quantification of the proportion of WBCs that were marked was achieved by comparing the intensity of the signal obtained from each sample to control DNA from HeLa cells that contained a single copy of the provirus that was then serially diluted in DNA extracted from nontransduced HeLa cells. Analysis of 1 μg template DNA (∼ 1.5 × 105 test cells) at each time point allowed the limit of gene-marked cells detectable to be reduced to close to 0.001% (assuming 1 vector copy per cell, dashed horizontal line). (B) Detection of different proviral integration sites in PB cells using LAM-PCR. The data indicate that multiple clones contributed to the early output of retrovirally marked WBCs in all 3 patients. M indicates a 100-bp DNA ladder; +C, positive control (0.02 ng DNA from monoclonal HeLa cells transduced with the same vector); and –C (1 μg nontransduced HeLa DNA), as well as –7 (DNA extracted from the PB of this patient taken 7 days before the transplantation was performed), negative controls.
