Clonal tracking analysis. (A) Rhesus macaque model: autologous transplantation of retrovirally transduced CD34⁺ cells. Animals were given G-CSF + SCF for 5 days, and mobilized PBSCs were collected by leukapheresis. Purified CD34⁺ cells were transduced with either G1Na or LNL6 standard retroviral marking vectors as previously described.^33,35  Transduced CD34⁺ cells were reinfused into the monkeys after 500 cGy × 2 total body irradiation. Animals were followed without intervention for at least 1 year before entry into the current cytokine treatment studies. (B) Schematic of the LAM-PCR methodology for identification of retroviral insertion sites. (i) Linear PCR by repeated primer extension from a biotinylated oligonucleotide and enrichment of the DNA product by capture with avidin-coated magnetic beads. (ii) Double-stranded DNA synthesis on the primer extension product by random hexanucleotide priming. (iii) DNA restriction digestion with TasI. (iv) Ligation of an asymmetric oligonucleotide ligation cassette (LC) to the overhanging sequence at the TasI digestion site. (v) Nested exponential PCR amplifications using primer pairs LCI-LTRII, LCII-LTRIII. (C) Final PCR amplification using a fluorescent primer allows separation and precise sizing of LAM-PCR products by way of comparison to size standards on an automated sequencer and analysis by using Gene Scan software. (D) Samples (100 ng) of a single DNA preparation from the granulocytes of an animal following transplantation with vector-transduced cells. The absolute number of independent clones detected by performance of each duplicate LAM-PCR procedure is shown. By the time 6 duplicates are run, the absolute clone number present is approached, and the mean clone number is calculated by counting the bands present in 6 duplicates on each sample run independently 3 separate times (for a total of 18 replicates on each sample). Error bars indicate the standard deviation for 3 independent experiments.