title: Using quantitative real-time PCR to determine donor cell engraftment in a competitive murine bone marrow transplantation model.
abstract: Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.
authors.affiliation: Division of Hematology-oncology, Department of Medicine, Medical University of South Carolina, USA.
authors.forename: Ningfei
authors.initials: N
authors.lastname: An
authors.forename: Yubin
authors.initials: Y
authors.lastname: Kang
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D004247:DNA / Q000032:analysis / Q000097:blood / Q000235:genetics; D015342:DNA Probes; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006085:Graft Survival / Q000502:physiology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D023421:Models, Animal; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014998:Y Chromosome
chemical_list: D015342:DNA Probes; D004247:DNA