| Research Abstract |
The present investigation was undertaken to develop a method to introduce a gene at high efficiency into hematopoletic stem cells. As the first step of study, we tried to make a retrovirus vector which is capable of expressing in stem cells. This was attained by replacing the enhancer of Molony leukemia LTR promoter of N2 vector with the enhancer of mutant polyoma virus. This vector, named W5, was used in the following experiments. Next, we selected a high producer of the packaging cell line for amphotropic virus. Original cultures of GPE86 cells, when transfected with W5, produced 3 x 10^6 PFC/ml of virus. After subcloning, we obtained a high producer M3, which were able to produce 3 x 10^7 PFU/ml of the virus.
Murine bone marrow cells were cultured on the monolayer of bone marrow stromal cell line PA6-neo. To this co-culture, various combinations of cytokines were added. Dexter culture system was also used. We found that combination of PA6-neo and IL-1, 3, 6 plus SCF as well as Dexter culture system were the best to maintain stem cells. In these culture systems, W5 vector was infected to bone marrow cells by transwell culture with M3, and the recovered cells were introvenously injected into 95OR irradiated mice. The presence of neo gene in splenic colonies was shown by PCR and Southern methods. These results strongly suggested that the neo gene was successfully introduced into hemopoietic stem cells.
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