Figure 4.
Figure 4. Schematic summary of the detection of individual clones contributing to granulocyte production before and after 3 Gy TBI. (A) The left-hand panel shows results from animal 96E019 and right-hand panel results from animal 96E025, and includes all clones detected at least once in a total of 18 separate LAM-PCR reactions (3 independent sets of 6 replicates). ▪ represents the detection of a specific clone at least once from a given time; □, no detection of a specific clone; and ↓, the time of 3 Gy TBI. Each column represents a separate time point: animal 96E019 6 months after transplantation, 7 months after transplantation, 9 months after transplantation, 1 year after transplantation, 1 year 11 months after transplantation, 2 years after transplantation, 1 week after 3 Gy TBI, 1 month after 3 Gy TBI, 2 months after 3 Gy TBI, 4 months after 3 Gy TBI, 5 months after 3 Gy TBI, 6 months after 3 Gy TBI, 9 months after 3 Gy TBI, 1 year after 3 Gy TBI; animal 96E025 7 months after transplantation, 9 months after transplantation, 1 year after transplantation, 3 years after transplantation, 1 week after 3 Gy TBI, 2 weeks after 3 Gy TBI, 3 weeks after 3 Gy TBI, 1 month after 3 Gy TBI, 2 months after 3 Gy TBI, 3 months after 3 Gy TBI, 4 months after 3 Gy TBI, 5 months after 3 Gy TBI, 6 months after 3 Gy TBI, 7 months after 3 Gy TBI, 8 months after 3 Gy TBI, 9 months after 3 Gy TBI, 10 months after 3 Gy TBI, 11 months after 3 Gy TBI, 1 year after 3 Gy TBI, 1 year 2 months after 3 Gy TBI, 1 year 3 months after 3 Gy TBI, 1 year 4 months after 3 Gy TBI. The arrows indicate the time of 3 Gy TBI. Numbering above panels refers to time in months before and after 3 Gy TBI. (B) Use of insertion-specific PCR tracking primers to verify presence or absence of individual clones. The arrow indicates the time of 3 Gy TBI, 3 years after initial transplantation in animal 96E025. There were 4 insertion-specific primers designed to amplify individual insertion site clones from animal 96E025, initially identified from LAM-PCR on the pre-TBI sample. Each insertion-specific primer was used along with an LTR primer to amplify an individual insertion. Those clones chosen included 3q26.2 within the mds1 gene, 18q21.2 within the dcc gene, 1p36.13 within a repeat element, and 18q21.31 within the hak gene. The time points for amplification were 1 (1 year after transplantation); 2 (2 years 10 months after transplantation); 3 (3 weeks after 3 Gy TBI); 4 (2 months after 3 Gy TBI); 5 (3 months after 3 Gy TBI); 6 (7 months after 3 Gy TBI); 7 (10 months after 3 Gy TBI); 8 (1 year after 3 Gy TBI); 9 (negative control normal rhesus macaque DNA). This clone-specific PCR confirms the clonal instability seen by LAM-PCR, with many clones disappearing and reappearing, and a minority not detectable at the most recent follow-up, possibly indicating complete deletion. (C) Semiquantitative analysis of the level of contribution of clones 3q26.2 (straight line) and 1p36.13 (dashed line) to granulocytes from animal 96E025. The arrow indicates the time of 3 Gy TBI. The relative clonal contribution was normalized against β-actin, and the first time point (3 months before 3 Gy TBI) was chosen as a baseline level. By 6 and 12 months after TBI, the relative contribution to granulopoiesis from these clones was stable and not significantly changed compared with baseline.

Schematic summary of the detection of individual clones contributing to granulocyte production before and after 3 Gy TBI. (A) The left-hand panel shows results from animal 96E019 and right-hand panel results from animal 96E025, and includes all clones detected at least once in a total of 18 separate LAM-PCR reactions (3 independent sets of 6 replicates). ▪ represents the detection of a specific clone at least once from a given time; □, no detection of a specific clone; and ↓, the time of 3 Gy TBI. Each column represents a separate time point: animal 96E019 6 months after transplantation, 7 months after transplantation, 9 months after transplantation, 1 year after transplantation, 1 year 11 months after transplantation, 2 years after transplantation, 1 week after 3 Gy TBI, 1 month after 3 Gy TBI, 2 months after 3 Gy TBI, 4 months after 3 Gy TBI, 5 months after 3 Gy TBI, 6 months after 3 Gy TBI, 9 months after 3 Gy TBI, 1 year after 3 Gy TBI; animal 96E025 7 months after transplantation, 9 months after transplantation, 1 year after transplantation, 3 years after transplantation, 1 week after 3 Gy TBI, 2 weeks after 3 Gy TBI, 3 weeks after 3 Gy TBI, 1 month after 3 Gy TBI, 2 months after 3 Gy TBI, 3 months after 3 Gy TBI, 4 months after 3 Gy TBI, 5 months after 3 Gy TBI, 6 months after 3 Gy TBI, 7 months after 3 Gy TBI, 8 months after 3 Gy TBI, 9 months after 3 Gy TBI, 10 months after 3 Gy TBI, 11 months after 3 Gy TBI, 1 year after 3 Gy TBI, 1 year 2 months after 3 Gy TBI, 1 year 3 months after 3 Gy TBI, 1 year 4 months after 3 Gy TBI. The arrows indicate the time of 3 Gy TBI. Numbering above panels refers to time in months before and after 3 Gy TBI. (B) Use of insertion-specific PCR tracking primers to verify presence or absence of individual clones. The arrow indicates the time of 3 Gy TBI, 3 years after initial transplantation in animal 96E025. There were 4 insertion-specific primers designed to amplify individual insertion site clones from animal 96E025, initially identified from LAM-PCR on the pre-TBI sample. Each insertion-specific primer was used along with an LTR primer to amplify an individual insertion. Those clones chosen included 3q26.2 within the mds1 gene, 18q21.2 within the dcc gene, 1p36.13 within a repeat element, and 18q21.31 within the hak gene. The time points for amplification were 1 (1 year after transplantation); 2 (2 years 10 months after transplantation); 3 (3 weeks after 3 Gy TBI); 4 (2 months after 3 Gy TBI); 5 (3 months after 3 Gy TBI); 6 (7 months after 3 Gy TBI); 7 (10 months after 3 Gy TBI); 8 (1 year after 3 Gy TBI); 9 (negative control normal rhesus macaque DNA). This clone-specific PCR confirms the clonal instability seen by LAM-PCR, with many clones disappearing and reappearing, and a minority not detectable at the most recent follow-up, possibly indicating complete deletion. (C) Semiquantitative analysis of the level of contribution of clones 3q26.2 (straight line) and 1p36.13 (dashed line) to granulocytes from animal 96E025. The arrow indicates the time of 3 Gy TBI. The relative clonal contribution was normalized against β-actin, and the first time point (3 months before 3 Gy TBI) was chosen as a baseline level. By 6 and 12 months after TBI, the relative contribution to granulopoiesis from these clones was stable and not significantly changed compared with baseline.

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