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Drug resistance genes such as those coding for a methotrexate-resistant dihydrofolate reductase (DHFR) or the thymidine kinase from herpes simplex virus can be used to confer a proliferative advantage on bone marrow cells of mice. As a result of this proliferative advantage, transformed cells become the predominant population in the bone marrow. Efficient gene expression was obtained for both the thymidine kinase and DHFR genes inserted into mouse bone marrow. Such gene insertion techniques may ultimately lead to the cure of life-threatening globinopathies such as sickle cell disease or the beta thalassemias. They may also be useful in reducing the hematopoietic toxicity of anticancer drugs.

DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene sequences.

DNA-mediated gene transfer was used to introduce DNA from a methotrexate-resistant mouse fibroblast cell line into mouse bone marrow cells. This cell line contained a methotrexate-resistant dihydrofolate reductase, active at 10(-4) M methotrexate, which was electrophoretically separable from the wild-type mouse enzyme. Transformed hematopoietic cells were returned to irradiated mice and selected in vivo by methotrexate administration. Some recipients of transformed marrow cells expressed the electrophoretically distinct, methotrexate-resistant dihydrofolate reductase in hematopoietic cells. These observations suggest that successful transformation of marrow stem cells to methotrexate resistance is accomplished by insertion of a dihydrofolate reductase gene coding for a mutant enzyme that is highly resistant to methotrexate.

Resistance to methotrexate was induced in bone marrow cells of mice by transformation in vitro with DNA from a drug-resistant cell line. Transformed cells were injected in vivo and haematopoietic cells expressing resistance were selected by drug treatment of recipients. Transformed cells had elevated levels of dihydrofolate reductase and demonstrated a proliferative advantage over untransformed cells, indicating successful gene transfer.