Individually picked CFU-GM colonies analyzed by LAM-PCR reveal this mouse was repopulated by a single repopulating cell that possessed 5 individual vector integrants
| Mouse 7.23 Colony number . | Integration site no. . | hu β-actin . | ||||
|---|---|---|---|---|---|---|
| 1 . | 2 . | 3 . | 4 . | 5 . | ||
| 7.23-44 | + | + | + | + | + | + |
| 7.23-47 | + | + | + | + | + | − |
| 7.23-63 | + | + | + | + | (+) | + |
| 7.23-68 | + | + | + | + | + | + |
| 7.23-69 | + | + | + | + | + | (+) |
| 7.23-85 | (+) | + | + | + | − | + |
| 7.23-87 | + | + | + | + | + | − |
| 7.23-88 | + | + | + | + | + | + |
| 7.23-90 | + | + | + | + | + | + |
| 7.23-91 | + | + | + | + | + | + |
| 7.23-92 | − | − | + | + | − | − |
| 7.23-93 | + | + | + | + | + | + |
| 7.23 total bone marrow | + | + | + | + | + | + |
| Nontransplanted BM | − | − | − | − | − | − |
| MOCK-transduced colony | − | − | − | − | − | + |
| Mouse 7.23 Colony number . | Integration site no. . | hu β-actin . | ||||
|---|---|---|---|---|---|---|
| 1 . | 2 . | 3 . | 4 . | 5 . | ||
| 7.23-44 | + | + | + | + | + | + |
| 7.23-47 | + | + | + | + | + | − |
| 7.23-63 | + | + | + | + | (+) | + |
| 7.23-68 | + | + | + | + | + | + |
| 7.23-69 | + | + | + | + | + | (+) |
| 7.23-85 | (+) | + | + | + | − | + |
| 7.23-87 | + | + | + | + | + | − |
| 7.23-88 | + | + | + | + | + | + |
| 7.23-90 | + | + | + | + | + | + |
| 7.23-91 | + | + | + | + | + | + |
| 7.23-92 | − | − | + | + | − | − |
| 7.23-93 | + | + | + | + | + | + |
| 7.23 total bone marrow | + | + | + | + | + | + |
| Nontransplanted BM | − | − | − | − | − | − |
| MOCK-transduced colony | − | − | − | − | − | + |
The 5 vector/human genome junction bands shown in Figure 2 could be seen in virtually all individually picked CFU-GM colonies derived from the NOD/SCID mouse. The same 5 bands could be detected in the total bone marrow of this mouse. The results of this analysis reveal that this mouse was repopulated by a single SRC that gave rise to virtually all the hematopoietic cells in this mouse. In some cases when a particular band was barely visible, colony DNA was analyzed by PCR tracking, where the known sequences of the vector and specific site in the human genome (Table 3) can be used to generate primers and allow for more sensitive detection of the presence of the band.16 PCR for human β-actin is used as a control. (+) denotes weak presence of amplified product from PCR tracking analysis.