title: High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.
abstract: 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)
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, Rockville, MD 20550, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: R P
authors.initials: RP
authors.lastname: Wersto
authors.forename: J A
authors.initials: JA
authors.lastname: Allay
authors.forename: B A
authors.initials: BA
authors.lastname: Agricola
authors.forename: M E
authors.initials: ME
authors.lastname: Metzger
authors.forename: A W
authors.initials: AW
authors.lastname: Nienhuis
authors.forename: D A
authors.initials: DA
authors.lastname: Persons
authors.forename: B P
authors.initials: BP
authors.lastname: Sorrentino
mesh_terms: D000818:Animals; D016207:Cytokines / Q000494:pharmacology*; D018076:DNA, Complementary / Q000008:administration & dosage; D005353:Fibronectins / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology*; D049452:Green Fluorescent Proteins; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D016131:Lymphocyte Subsets / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D008253:Macaca mulatta; D010446:Peptide Fragments / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods*
chemical_list: D016207:Cytokines; D018076:DNA, Complementary; D005353:Fibronectins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase