title: Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells.
abstract: The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications.
authors.affiliation: 1 Discovery Genomics, Inc. , Minneapolis, Minnesota.
authors.forename: Kendra A
authors.initials: KA
authors.lastname: Hyland
authors.affiliation: 1 Discovery Genomics, Inc. , Minneapolis, Minnesota.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Olson
authors.affiliation: 1 Discovery Genomics, Inc. , Minneapolis, Minnesota.|2 Department of Genetics, Cell Biology and Development, University of Minnesota , Minneapolis, Minnesota.
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
mesh_terms: D018952:Antigens, CD34 / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004176:Dipyridamole / Q000008:administration & dosage; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells; D006801:Humans; D008727:Methotrexate / Q000008:administration & dosage; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D004176:Dipyridamole; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate