Determination of donor-type chimerism using a semi-quantitative PCR-based method in a canine model for bone marrow transplantation.
Dogs are used in preclinical transplantation models to study methods of allogeneic bone marrow transplantation (BMT). The evaluation of chimerism is of major significance for the investigation of graft-vs.-host (GvH) and host-vs.-graft (HvG) reactions. To detect and quantitate male donor cells after a sex-mismatched (male to female) allogeneic BMT, we established a semi-quantitative polymerase chain reaction (PCR) assay. Based on the canine Y-chromosome sex-determining region (Sry) sequence, we designed primer specific for the detection of male DNA and optimised PCR conditions and cycle numbers. Artificial mixtures of male and female leukocytes were used to analyse the sensitivity of the assay. To validate our established method, we determined the percentage of chimerism in three transplanted female dogs. Under optimised conditions, the established PCR assay specifically detected male cells down to 0.01%, which corresponds to 0.1ng of transplanted male DNA. The percentage of chimerism could be quantitated either by agarose gel analysis or Southern blot analysis. Using our assay, we could confirm the percentage of chimerism in blood samples of three transplanted female canines, previously determined by karyotype analysis as 0, 100 and 100%, respectively. The established semi-quantitative PCR assay offers a quick, simple, accurate and sensitive way of evaluating and quantitating the percentage of chimerism in a sex-mismatched canine BMT model.
Fiegler
Heike
H
GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Klinische Molekularbiologie und Tumorgenetik, 81377, München, Germany.
Knabel
Michael
M
Franz
Monika
M
Kolb
Hans Jochem
HJ
Just
Ursula
U
D000818:Animals; D016026:Bone Marrow Transplantation; D002678:Chimera; D004285:Dogs; D005260:Female; D024282:Genes, sry; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity