LONG-TERM PROTECTION OF RECIPIENT MICE FROM LETHAL DOSES OF METHOTREXATE BY MARROW INFECTED WITH A DOUBLE-COPY VECTOR RETROVIRUS CONTAININGA MUTANT DIHYDROFOLATE-REDUCTASE

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEO(r) gene and a mutated dihydrofolate reductase cDNA (Leu 22 --> Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Prima ry, secondary, and even tertiary recipients transplanted with bone mar row cells infected with the recombinant virus showed protection from l ethal methotrexate toxicity. The viral construct containing a SV-40 pr omoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deami nase promoter, although both afforded protection. Evidence for integra tion into blood cells of both the NEO(r) gene and the mutated dihydrof olate reductase gene was obtained by polymerase chain reaction; sequen cing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoieti c progenitor cells in the mouse can be successfully transduced with a drug resistance gene.