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Purpose: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells., Method: In this study, we investigate retroviral transfer of cytosolic 5'-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2'-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment., Results: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression., Conclusion: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker.