Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors

Naoko Takebe, Saori Nakahara, Shi Cheng Zhao, Debasis Adhikari, Ali U. Ural, Marian Iwamoto, Debabrata Banerjee, Joseph Bertino

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.

Original languageEnglish (US)
Pages (from-to)910-919
Number of pages10
JournalCancer gene therapy
Volume7
Issue number6
DOIs
StatePublished - Jan 1 2000
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Methotrexate
Complementary DNA
Hematopoietic Stem Cells
Serine
Messenger RNA
Cytoprotection
Transgenes
Northern Blotting
Real-Time Polymerase Chain Reaction
Enzymes
Genes
Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Cancer Research

Cite this

Takebe, Naoko ; Nakahara, Saori ; Zhao, Shi Cheng ; Adhikari, Debasis ; Ural, Ali U. ; Iwamoto, Marian ; Banerjee, Debabrata ; Bertino, Joseph. / Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors. In: Cancer gene therapy. 2000 ; Vol. 7, No. 6. pp. 910-919.
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Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors. / Takebe, Naoko; Nakahara, Saori; Zhao, Shi Cheng; Adhikari, Debasis; Ural, Ali U.; Iwamoto, Marian; Banerjee, Debabrata; Bertino, Joseph.

In: Cancer gene therapy, Vol. 7, No. 6, 01.01.2000, p. 910-919.

Research output: Contribution to journalArticle

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