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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 high-titer 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.

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Acknowledgements

We gratefully acknowledge the assistance of Diane Domingo for FACS analysis, Jessica Boklan for providing optimal conditions for colony PCR, and Emine Ercikan-Abali for providing the Pkt7 plasmid. Supported by CA59350, CA08748, and CA09512 (NT).

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Correspondence to Malcolm AS Moore.

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Takebe, N., Xu, LC., MacKenzie, K. et al. Methotrexate selection of long-term culture initiating cells following transduction of CD34+ cells with a retrovirus containing a mutated human dihydrofolate reductase gene. Cancer Gene Ther 9, 308–320 (2002). https://doi.org/10.1038/sj.cgt.7700443

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