vector-producing packaging cells followed by incubation of marrow cells in a vector- 
containing long-term marrow culture system for 4 days. Transduced autologous marrow 
was infused into otherwise lethally irradiated (9.2 Gy TBI) dogs, and reconstituted 
marrow was examined for the presence and expression of retrovirus vectors. Six dogs 
received 0.4 - 1.0 x 10 8 marrow cells/kg transduced with retrovirus vectors which 
contained both the neo gene and the human ADA gene. Four of six dogs engrafted. 
Their marrows have shown 1-20% G418-resistant CFU-GM colonies intermittently for 
more than two years after transplantation (Table 1). G418-resistant colonies were not 
observed in marrows from control dogs. 
Culture results were confirmed by polymerase chain reaction (PCR) demonstrating the 
presence of the neo gene in marrow cells so far more than three years after 
transplantation (Figures 1 and 2 and Table 2). 
The neo gene was also found by PCR in peripheral blood and lymph node lymphocytes 
and peripheral blood granulocytes so far up to more than three years posttransplant 
(Figures 1 and 2 and Table 2). PCR analysis of those samples of marrow cells, 
peripheral blood granulocytes and peripheral blood and lymph node lymphocytes which 
had been shown to contain the neo gene also detected the gene for human ADA (Figure 
3). 
However, repeated analysis of peripheral blood and marrow cells for human ADA gene 
expression by starch gel electrophoresis was negative. This experiment demonstrates that 
transduction of marrow by cocultivation and long-term marrow culture leads to long-term 
persistence of the retrovirus vector in myeloid and lymphoid cells, strongly suggesting 
transduction of long-term repopulating stem cells. For currently unknown reasons, long- 
term expression of the neo gene but not the human ADA gene was observed. 
In a different experimental approach, autologous marrow, obtained 7 days after 
cyclophosphamide treatment (40 mg/kg once i.v.) at the time of the peripheral blood 
neutrophil nadir, was cocultivated for 24 hours on vector-producing cells and reinfused 
(0.06 - 0.18 x 10 8 cells/kg) into four irradiated (9.2 Gy TBI) dogs. Two dogs engrafted 
and showed 1-10% G418-resistant CFU-GM colonies intermittently for so far more than 
two years, similar to the surviving dogs of the previous experiment (Tables 2 and 3). 
Marrow from control dogs did not show G418-resistant colonies. 
PCR analysis of marrow cells, peripheral blood granulocytes and peripheral blood and 
lymph node lymphocytes obtained so far more than two years after marrow 
transplantation demonstrated the presence of the neo gene (Table 2). Similar to the 
previous results, no expression of human ADA was detected even though the human 
ADA gene was present in cells containing the neo gene (Figure 3). Thus, this 
experiment shows that cocultivation of marrow obtained from dogs after treatment with 
cyclophosphamide leads to long-term persistence of retrovirus vectors in myeloid and 
lymphoid cells, providing evidence for gene transduction into long-term repopulating 
stem cells. For unknown reasons, only the neo gene but not the human ADA gene was 
expressed long-term. 
Dilution experiments with cells from the dogs indicate that the fraction of marrow cells, 
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Recombinant DNA Research, Volume 18 
