Selective expression of methotrexate-resistant dihydrofolate reductase (DHFR) activity in mice transduced with DHFR retrovirus and administered methotrexate

To determine the effect of in vivo pharmacological selective pressure on the insertion and expression of new gene sequences, retroviral-mediated transfer of a methotrexate-resistant dihydrofolate reductase (Mtx(r)-DHFR) gene in hematopoietic tissue was investigated using a murine syngeneic bone marrow transplant system. A series of recombinant retroviral vectors were constructed to contain long terminal repeat (LTR) regions from different murine retroviruses associated with various proliferative disorders of the lymphohematopoietic system, including Moloney leukemia virus, spleen focus- forming virus (anemia strain) and myeloproliferative sarcoma virus. High- titer DHFR virus (10⁷ colony-forming units/ml) was generated by gene amplification, adapting virus-producer cell lines to grow in medium containing increasing concentrations of Mtx. This high-titer DHFR virus was used to introduce the Mtx(r)-DHFR gene into murine hematopoietic tissue by injecting DHFR virus-exposed marrow into lethally irradiated syngeneic recipient mice that subsequently were administered Mtx. Southern blot analysis of spleen DNA demonstrated insertion of the DHFR provirus in all surviving mice transplanted with DHFR virus-exposed marrow. However, enzymatic assay of crude spleen extracts demonstrated the presence of Mtx(r)- DHFR activity only in mice that were administered Mtx; nonadministered animals or animals transplanted with control (neo) virus-infected marrow contained undetectable drug-resistant enzyme activity. These results suggest the selective outgrowth of hematopoietic tissue harboring and expressing a DHFR provirus in animals administered Mtx and have implications for the application of drug-resistance gene insertion in somatic tissues of animals and humans.