Efficient expression of functional human MDR1 gene in murine bone marrow
after retroviral transduction of purified hematopoietic stem cells
T Licht, I Aksentijevich, MM Gottesman and I Pastan
Laboratory of Molecular Biology, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892-4255, USA.
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.
Volume 86,
Issue 1,
pp. 111-121,
07/01/1995
Copyright © 1995 by The American Society of Hematology
