Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Abstract

SHORT ABSTRACT Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions. LONG 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.

Cox-2 inhibition potentiates mouse bone marrow stem cell engraftment and differentiation-mediated wound repair

“…Expression of Y-linked zinc finger protein-1 (Zfy-1) along with Bcl-2 gene as internal control was quantified using qPCR analysis in the regenerated wound tissue of female mice as described earlier [16]. Standard curve was plotted using 2-δCt reading from known male/female DNA standard mixtures [21]. Cell engraftment was represented as % male DNA plotted on y-axis (mean of 2-δCt value of triplicates to the known % male DNA in the mixture with linear regression fitting).…”

Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice

“…At 3 months post transplantation, the secondary transplant mice receiving Pim TKO cells had significantly reduced peripheral white blood cell counts (Figure 4A). Donor cell engraftment in BM was quantified at 4 months post transplantation using a quantitative PCR-based method measuring the sex-determining region Y (Zfy1) [13,16]. We found that the male donor cells engraftment in secondary Pim TKO transplant recipients was significantly lower than that in controls (Figure 4B).…”

“…In the absence of serial transplant experiments, this finding may lead investigators to assume that HSC population is unaltered in Pim-deficient mice. The PCR-based method that we reported recently [13] allows us to reliably determine donor cell engraftment in our transplant experiments.…”

“…We recently reported a quantitative real-time PCR-based technique for determination of donor cell engraftment in a competitive murine transplantation model [13]. Our PCR method measures the Y chromosome specific gene, i.e., Zfy-1, and can be used for any strain of mouse transplantation models.…”

Mechanical Loading Attenuates Radiation-Induced Bone Loss in Bone Marrow Transplanted Mice

“…Samples were vortexed, then centrifuged to separate powdered mineral, and aspirate was processed in spin columns to isolate DNA according to Qiagen’s instructions. Quantitative PCR for both the GFP transgene and male Y chromosome-specific Zfy1 [40] was then carried out in triplicate with the QuantiTect SYBR-Green PCR kit (Qiagen, Valencia, CA) and normalized to ApoB . Murine-specific primers are specified in Table 1.…”