American Society of Hematology

Figure 3.

$Figure 3. Sex-mismatch bone marrow transplantation model. (A) Flow chart of the experimental procedure. Unfractionated BM cells from male Balb/c donor mice were transplanted into lethally irradiated syngenic female recipients. After hematopoietic recovery, the efficiency of engraftment was evaluated by competitive PCR by quantifying the copies of donor-specific Y-chromosome sequences in the bone marrow of the animals that received a transplant, using β-globin as reference gene. (B) Schematic representation of the templates for quantitative PCR. Amplification of the Y-chromosome sequence and of the β-globin gene were obtained with primer pairs Y-F/Y-R and BG-F/BG-R, respectively. The multicompetitor used for competitive PCR contains a core sequence of 218 bp, corresponding to a 20-bp–deleted version of the mouse β-globin amplification fragment, flanked by Y-F and Y-R primer sequences. (C) Example of competitive PCR amplification. Fixed amounts of sample DNA from PBMCs were mixed with scalar amounts of the multicompetitor DNA and PCR amplified with the 2 primer pairs. After amplification, the gels were stained with ethidium bromide and the competitor (Comp), Y chromosome (Y chr), or β-globin DNA bands were quantified. According to the principles of competitive PCR, the ratio between the amplification products in each reaction is linearly correlated with the input DNA amounts for the 2 DNA species. Dashed red boxes indicate the point of equivalence. (D) Examples of FISH analysis on muscle sections from mice that received a transplant at 30 days after injection of AAV-VEGF, using a mouse-specific Y-chromosome probe labeled with FITC. The green dots, indicated by arrows in the enlargements, correspond to Y-chromosome–specific signals. Red indicates nuclei stained by propidium iodide; and M, muscle fibers. Images in panel D were obtained using a Zeiss LSM510 confocal microscope (Carl Zeiss, Göttingen, Germany), equipped with an Axiovert 100M reverse microscope and 40 ×/0.75 NA and 100 ×/1.30 NA oil objectives. LSM510 software 3.2 was used for image acquisition. Pictures were assembled with Adobe Photoshop 7.0 software.$

Sex-mismatch bone marrow transplantation model. (A) Flow chart of the experimental procedure. Unfractionated BM cells from male Balb/c donor mice were transplanted into lethally irradiated syngenic female recipients. After hematopoietic recovery, the efficiency of engraftment was evaluated by competitive PCR by quantifying the copies of donor-specific Y-chromosome sequences in the bone marrow of the animals that received a transplant, using β-globin as reference gene. (B) Schematic representation of the templates for quantitative PCR. Amplification of the Y-chromosome sequence and of the β-globin gene were obtained with primer pairs Y-F/Y-R and BG-F/BG-R, respectively. The multicompetitor used for competitive PCR contains a core sequence of 218 bp, corresponding to a 20-bp–deleted version of the mouse β-globin amplification fragment, flanked by Y-F and Y-R primer sequences. (C) Example of competitive PCR amplification. Fixed amounts of sample DNA from PBMCs were mixed with scalar amounts of the multicompetitor DNA and PCR amplified with the 2 primer pairs. After amplification, the gels were stained with ethidium bromide and the competitor (Comp), Y chromosome (Y chr), or β-globin DNA bands were quantified. According to the principles of competitive PCR, the ratio between the amplification products in each reaction is linearly correlated with the input DNA amounts for the 2 DNA species. Dashed red boxes indicate the point of equivalence. (D) Examples of FISH analysis on muscle sections from mice that received a transplant at 30 days after injection of AAV-VEGF, using a mouse-specific Y-chromosome probe labeled with FITC. The green dots, indicated by arrows in the enlargements, correspond to Y-chromosome–specific signals. Red indicates nuclei stained by propidium iodide; and M, muscle fibers. Images in panel D were obtained using a Zeiss LSM510 confocal microscope (Carl Zeiss, Göttingen, Germany), equipped with an Axiovert 100M reverse microscope and 40 ×/0.75 NA and 100 ×/1.30 NA oil objectives. LSM510 software 3.2 was used for image acquisition. Pictures were assembled with Adobe Photoshop 7.0 software.

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