Engraftment, Transduction, and Long-Term Clonogenic Capacity of Human CD34+CD38− Cells Transduced on Days 1, 2, and 3 Versus 5, 6, and 7
| Transduction Condition . | % CD45+ in Marrow . | No. G418R CFU . | Total HumanSpecfic CFU . | % G418 Resistance . | Result of PCR for Neo Gene . | No. Integrants: T-Cell Clones† . | No. Integrants: Myeloid Clones . |
|---|---|---|---|---|---|---|---|
| Days 1, 2, 3 - FL | 0.9 | 0 | 10 | 0 | − | − | − |
| 1.5 | 0 | 33 | 0 | − | − | − | |
| 2.6 | 0 | 53 | 0 | − | − | − | |
| 0 | 0 | 0 | 0 | − | − | − | |
| 2.4 | 0 | 83 | 0 | − | − | − | |
| 3.7 | 0 | 30 | 0 | − | − | − | |
| Average = 1.9 ± 0.6 | 0 | 34.8 ± 13.3 | 0 | Total = 0/6 | |||
| Days 5, 6, 7 - FL* | 0 | 0 | 0 | 0 | − | − | − |
| 0.1 | 0 | 3 | 0 | − | − | − | |
| 0 | 0 | 0 | 0 | − | − | − | |
| 0.2 | 0 | 6 | 0 | − | − | − | |
| Average = 0.1 ± 0.1 | 0 | 2.3 ± 1.4 | 0 | Total = 0/4 | |||
| Days 1, 2, 3 + FL | 3.1 | 0 | 193 | 0 | − | − | − |
| 2.8 | 0 | 123 | 0 | − | − | − | |
| 1.4 | 0 | 44 | 0 | − | − | − | |
| 7.3 | 0 | 336 | 0 | − | − | − | |
| 3.2 | 0 | 27 | 0 | − | − | − | |
| 0.8 | 0 | 3 | 0 | − | − | − | |
| Average = 3.1 ± 1.0 | 0 | 121 ± 56.3 | 0 | Total = 0/6 | |||
| Days 5, 6, 7 + FL* | 3.0 | 7 | 293 | 2.4 | + | 2 | 3 |
| 1.9 | 6 | 155 | 3.9 | + | 2 | 1 | |
| 5.2 | 0 | 216 | 0 | − | − | − | |
| 2.5 | 0 | 187 | 0 | − | − | − | |
| Average = 3.2 ± 0.7 | 3.3 | 212.8 ± 29.5 | 1.6 | Total = 2/4 | |||
| Transduction Condition . | % CD45+ in Marrow . | No. G418R CFU . | Total HumanSpecfic CFU . | % G418 Resistance . | Result of PCR for Neo Gene . | No. Integrants: T-Cell Clones† . | No. Integrants: Myeloid Clones . |
|---|---|---|---|---|---|---|---|
| Days 1, 2, 3 - FL | 0.9 | 0 | 10 | 0 | − | − | − |
| 1.5 | 0 | 33 | 0 | − | − | − | |
| 2.6 | 0 | 53 | 0 | − | − | − | |
| 0 | 0 | 0 | 0 | − | − | − | |
| 2.4 | 0 | 83 | 0 | − | − | − | |
| 3.7 | 0 | 30 | 0 | − | − | − | |
| Average = 1.9 ± 0.6 | 0 | 34.8 ± 13.3 | 0 | Total = 0/6 | |||
| Days 5, 6, 7 - FL* | 0 | 0 | 0 | 0 | − | − | − |
| 0.1 | 0 | 3 | 0 | − | − | − | |
| 0 | 0 | 0 | 0 | − | − | − | |
| 0.2 | 0 | 6 | 0 | − | − | − | |
| Average = 0.1 ± 0.1 | 0 | 2.3 ± 1.4 | 0 | Total = 0/4 | |||
| Days 1, 2, 3 + FL | 3.1 | 0 | 193 | 0 | − | − | − |
| 2.8 | 0 | 123 | 0 | − | − | − | |
| 1.4 | 0 | 44 | 0 | − | − | − | |
| 7.3 | 0 | 336 | 0 | − | − | − | |
| 3.2 | 0 | 27 | 0 | − | − | − | |
| 0.8 | 0 | 3 | 0 | − | − | − | |
| Average = 3.1 ± 1.0 | 0 | 121 ± 56.3 | 0 | Total = 0/6 | |||
| Days 5, 6, 7 + FL* | 3.0 | 7 | 293 | 2.4 | + | 2 | 3 |
| 1.9 | 6 | 155 | 3.9 | + | 2 | 1 | |
| 5.2 | 0 | 216 | 0 | − | − | − | |
| 2.5 | 0 | 187 | 0 | − | − | − | |
| Average = 3.2 ± 0.7 | 3.3 | 212.8 ± 29.5 | 1.6 | Total = 2/4 | |||
Bone marrow was recovered from bnx mice after engraftment periods of 9 to 11 months. Human-specific colony-forming assays were plated from the bone marrow of each mouse (3 × 105bnx/hu bone marrow cells per sample) with and without addition of the selective agent G418 (0.9 mg/mL active). The extent of transduction was calculated as (no. G418-resistant/total human CFU) × 100 = % G418 resistance. Genomic DNA was isolated from samples of the bone marrow, and PCR to detect the presence of proviral integrants carrying the neo gene was performed. If positive by neo PCR, the marrow was further subjected to single-cell cloning and single-colony inverse PCR.
Two mice that had received human marrow CD34+CD38− cells cultured for 7 days with and without FL died of natural causes several months after the cotransplantation, and viable marrow could not be recovered for assays. The deaths were unrelated to receiving the human cell xenograft.
Samples in which no neo gene marking in total BM was detectable were not subjected to single-cell cloning and inverse PCR. These samples are shown as (−).