Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells.
A procedure for efficient transfer of the human MDR1 (multi-drug resistance) gene into murine hematopoietic stem cells was developed. Cells expressing Sca-1 but no lineage-specific or major histocompatibility complex (MHC) class II antigens (Lin-MHC II-Sca-1+) were enriched from 5-fluorouracil-pretreated bone marrow by Ficoll density-gradient and immunomagnetic sorting. Purified cells were cocultured with growth factors and fibroblasts producing replication-deficient retroviruses containing human MDR1 cDNA. Fluorescence-activated cell sorter analysis and rhodamine-123 efflux experiments showed that greater than 60% of cocultured hematopoietic cells expressed functional human P-glycoprotein. After 6 to 8 days, hematopoietic cells were injected intravenously into sublethally irradiated SCID mice. Stem cell properties of the isolated population were confirmed by sustained expression of MDR1 marker cDNA for greater than 4 to 6 months after transplantation, multilineage engraftment, and presence of MDR1 cDNA in bone marrow of secondary recipient mice after retransplantation. Reconstitution of H-2K-mismatched SCID mice showed high engraftment capacity of Lin-MHC II-Sca-1+ cells. MDR1 cDNA was detected in blood of 78% of recipients. P-glycoprotein was expressed in bone marrow of 71% of mice, in both lymphocytes and myelomonocytoid progenitors. P-glycoprotein function in host marrow was confirmed by rhodamine-123 efflux. Transduction of P-glycoprotein may be useful for gene therapy in two ways: to protect bone marrow from myelosuppression after chemotherapy and as a selectable marker in vivo for the introduction of otherwise nonselectable genes.
Licht
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Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Aksentijevich
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Gottesman
M M
MM
Pastan
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I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D000943:Antigens, Differentiation / Q000032:analysis; D001483:Base Sequence; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002478:Cells, Cultured / Q000637:transplantation; D018076:DNA, Complementary / Q000097:blood / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D005240:Feasibility Studies; D005822:Genetic Vectors / Q000235:genetics; D006183:H-2 Antigens / Q000235:genetics; D006251:Harvey murine sarcoma virus / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008969:Molecular Sequence Data; D011828:Radiation Chimera; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000943:Antigens, Differentiation; D018076:DNA, Complementary; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D011993:Recombinant Fusion Proteins