High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34+ cells

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vect...

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Bibliographic Details
Published in:Blood 2000-01, Vol.95 (2), p.445-452
Main Authors: DONAHUE, R. E, WERSTO, R. P, ALLAY, J. A, AGRICOLA, B. A, METZGER, M. E, NIENHUIS, A. W, PERSONS, D. A, SORRENTINO, B. P
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Biotechnology
Cytokines - pharmacology
DNA, Complementary - administration & dosage
Fibronectins - pharmacology
Fundamental and applied biological sciences. Psychology
Gene therapy
Granulocytes - cytology
Green Fluorescent Proteins
Health. Pharmaceutical industry
Hematopoietic Stem Cell Mobilization
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells - cytology
Hematopoietic Stem Cells - drug effects
Hematopoietic Stem Cells - physiology
Humans
Industrial applications and implications. Economical aspects
Luminescent Proteins - biosynthesis
Luminescent Proteins - genetics
Lymphocyte Subsets - immunology
Lymphocytes - cytology
Lymphocytes - immunology
Macaca mulatta
Peptide Fragments - pharmacology
Promoter Regions, Genetic
Recombinant Fusion Proteins - biosynthesis
Recombinant Proteins - pharmacology
Tetrahydrofolate Dehydrogenase - biosynthesis
Tetrahydrofolate Dehydrogenase - genetics
Transfection - methods
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Summary:We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.v95.2.445