Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction

Ming Xia Li, Debabrata Banerjee, Shi Cheng Zhao, Barry I. Schweitzer, Shin Mineishi, Eli Gilboa, Joseph R. Bertino

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66 Scopus citations


A double-copy Moloney leukemia virus-based retroviral construct containing both the Neo(R) gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock-infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR- amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX- resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

Original languageEnglish (US)
Pages (from-to)3403-3408
Number of pages6
Issue number11
StatePublished - Jun 1 1994


ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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