In vivo integration site analysis of primate leukocytes.
(A) LAM-PCR analysis in a rhesus macaque. DNA samples of 100 ng from FACS-sorted granulocytes (G) or mononuclear cells (MNCs) of rhesus macaque RC501 were analyzed at different time points after transplantation. Numbers indicate days after transplantation. M indicates a 100-bp DNA ladder; 100, 200, 300, 400, size of molecular weight marker in base pairs; C 0, negative control analysis on 1.4 μg nontransduced simian DNA; C 0.1, C 0.01, positive control analysis on 0.1 and 0.01 ng of DNA from a HeLa cell clone with a known single retrovirus insertion site corresponding to 15 and 1.5 genomic DNA equivalents, respectively; and H2O, water control. (B) LAM-PCR analysis in a baboon. DNA samples (100 ng) from FACS-sorted mononuclear cells (MNCs) of baboon T94433 were analyzed at different time points after transplantation. Note that the integration site signal is polyclonal throughout the observation period. Numbers indicate months after transplantation. M indicates a 100-bp DNA ladder; H2O, water control. (C) Reproducibility of LAM-PCR analysis in highly polyclonal rhesus macaque samples. From 4 to 6 repetitions of LAM-PCR analysis on individual 200-ng aliquots of DNA from purified granulocytes (G) obtained from rhesus macaques 95E003, 96E019, and RC706 were analyzed. Animal 95E003 was 40 months after transplantation, animal RC706 was 21 months after transplantation, and animal 96E019 was 27 months after transplantation at the time of sampling. For each animal, the separate amplifications are shown in adjacent lanes. M indicates a 50-bp DNA ladder.
