Methotrexate selection of long-term culture initiating cells following transduction of CD34+ cells with a retrovirus containing a mutated human dihydrofolate reductase gene Journal Article
| Authors: | Takebe, N.; Xu, L. C.; Mackenzie, K. L.; Bertino, J. R.; Moore, M. A. |
| Article Title: | Methotrexate selection of long-term culture initiating cells following transduction of CD34+ cells with a retrovirus containing a mutated human dihydrofolate reductase gene |
| Abstract: | A limitation of successful stem cell gene transfer to hematopoietic stem cells is low transduction efficiency. To overcome this hurdle and develop a gene transfer strategy that might be clinically feasible, retroviral vectors containing a drug resistance gene were utilized to transduce human CD34+-enriched cells and select gene-modified cells by drug administration. We constructed a hightiter retroviral vector containing a fusion gene (F/S-EGFP) consisting of a mutated dihydrofolate reductase (DHFR) (Leu22→Phe22, Phe31→Ser31; F/S) gene and enhanced green fluorescent protein (EGFP) cDNA. To test whether the fusion gene could function as a selectable marker, transduced CD34+ cells were assayed in long-term stromal co-cultures with and without addition of methotrexate (MTX). Without MTX exposure, the vector-transduced CD34+ cells generated 22-50% EGFP+ cobblestone area forming cells (CAFC) at week 5. By contrast, the vector-transduced cells cultured with MTX produced 96-100% EGFP+ CAFC in four separate experiments. These are the first investigations to demonstrate selection for transduced long-term culture initiating cells using MTX. The DHFR/MTX system holds promise for improving selection of gene-transduced hematopoietic progenitor cells in vivo. |
| Keywords: | human cell; mutation; methotrexate; flow cytometry; polymerase chain reaction; cd34 antigen; antimetabolites, antineoplastic; green fluorescent protein; stem cell transplantation; drug resistance, neoplasm; cell population; transfection; time; gene transfer; genetic transduction; genetic vectors; luminescent proteins; transduction, genetic; blotting, western; hybrid protein; recombinant fusion proteins; cell culture; fusion gene; gene therapy; hematopoietic stem cells; cell culture techniques; green fluorescent proteins; hematopoietic stem cell; dna primers; dihydrofolate reductase; tetrahydrofolate dehydrogenase; stroma cell; colony-forming units assay; antigens, cd34; cell selection; retrovirus; retroviridae; colony forming unit gm; virus vector; gene transfer techniques; enhanced green fluorescent protein; fusion protein; genes, viral; unidentified retrovirus; humans; human; priority journal; article; double mutant dihydrofolate reductase; stem cell selection |
| Journal Title: | Cancer Gene Therapy |
| Volume: | 9 |
| Issue: | 3 |
| ISSN: | 0929-1903 |
| Publisher: | Nature Publishing Group |
| Date Published: | 2002-03-01 |
| Start Page: | 308 |
| End Page: | 320 |
| Language: | English |
| DOI: | 10.1038/sj/cgt/7700443 |
| PUBMED: | 11896448 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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