Quantitative polymerase chain reaction assessment of chimerism in non-human primates after sex-mismatched islet and bone marrow transplantation.
BACKGROUND
Accurate assessment of chimerism in recipients of islet and bone marrow transplantation (BMT) may allow for a clearer assessment of the role of chimerism in islet engraftment or rejection. A quantitative polymerase chain reaction (PCR) assay was developed for the detection of the sex-determining region of the Y chromosome (SRY) in peripheral blood samples from female non-human primate recipients of allogeneic male islets and vertebral body marrow (VBM) from the same donor.


METHODS
The assay incorporates a synthetic internal standard (IS) containing the same primer template sequences as the target to compete for primer annealing and amplification. Each DNA sample was coamplified with a constant amount of IS. The concentration of male DNA in the test samples was calculated from the regression equation of a standard curve that was generated by plotting the logarithm of the ratio of the intensities of SRY to IS PCR products versus the logarithm of known percentages of input male DNA.


RESULTS
This method allows for a correction of the variability of efficiency of the PCR technique and also overcomes the drawback of time-consuming competitive PCR. Using this assay, we quantitated the amount of male DNA in samples taken from female baboon recipients of male islets and VBM. There was detectable male donor DNA in the samples taken one day after BMT; pre-BMT samples were negative. This technique works well for samples obtained from rhesus and cynomogus monkeys as well.


CONCLUSIONS
It is a practical method for accurately evaluation of chimerism after sex-mismatched allogeneic BMT in non-human primate models.
Han
D
D
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
Ricordi
C
C
Xu
X
X
Kenyon
N S
NS
D000818:Animals; D016026:Bone Marrow Transplantation; D002678:Chimera / Q000235:genetics*; D004247:DNA / Q000032:analysis; D005260:Female; D016381:Islets of Langerhans Transplantation; D007700:Kinetics; D008297:Male; D010215:Papio; D016133:Polymerase Chain Reaction; D011184:Postoperative Period; D019849:Sex Determination Processes; D014998:Y Chromosome / Q000235:genetics*
D004247:DNA