title: Mouse models of hematopoietic engraftment: limitations of transgenic green fluorescent protein strains and a high-performance liquid chromatography approach to analysis of erythroid chimerism.
abstract: Transgenic mouse strains ubiquitously expressing green fluorescent protein (GFP) have enabled investigators to develop in vivo transplant models that can detect donor contributions to many different tissues. However, most GFP transgenics lack expression of the reporter in the erythroid lineage. We evaluated expression of GFP in the bone marrow of the OsbY01 transgenic mouse (B6-GFP) in the context of CD71 and TER-119 expression and found that GFP fluorescence is lost prior to the basophilic erythroblast stage of development. However, platelets in B6-GFP mice were found to be uniformly positive for GFP. We therefore used the GFP transgenic model in combination with allelic variants of CD45 and the hemoglobin beta (Hbb) chain to develop a model system that allows all blood lineages to be followed in a mouse model of bone marrow transplantation (BMT). To detect Hbb variant molecules, we developed a new protocol based on high-performance liquid chromatography that is sensitive and precise, allowing rapid and quantitative analysis of erythroid chimerism. Platelet and leukocyte engraftment were detected by flow cytometry. BMT into sublethally irradiated (4 Gy) recipients demonstrated the failure of B6-GFP-derived cells to engraft relative to B6-CD45(a)-derived cells, suggesting that an immune barrier may prevent efficient engraftment of the transgenic cells in a setting of minimal ablation. These results establish limitations in the use of transgenic GFP expression as a donor marker in transplantation models.
authors.affiliation: Department of Medicine, University of Utah, 30 N 1900 East, Salt Lake City, 84132-2408, USA. gspangrude@mac.com
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Spangrude
authors.forename: Scott
authors.initials: S
authors.lastname: Cho
authors.forename: Otto
authors.initials: O
authors.lastname: Guedelhoefer
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Vanwoerkom
authors.forename: William H
authors.initials: WH
authors.lastname: Fleming
mesh_terms: D000483:Alleles; D000818:Animals; D001773:Blood Cells / Q000378:metabolism; D016026:Bone Marrow Transplantation / Q000379:methods*; D019070:Cell Lineage; D046528:Chimerism; D002851:Chromatography, High Pressure Liquid; D041905:Erythroid Cells / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D006454:Hemoglobins / Q000235:genetics; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D023421:Models, Animal; D010641:Phenotype; D019076:Transgenes / Q000235:genetics*
chemical_list: D006454:Hemoglobins; D049452:Green Fluorescent Proteins; D017493:Leukocyte Common Antigens