Abstract
Canine models are successfully applied to the study of haematopoietic stem cell transplantation (HSCT). Monitoring of haematopoietic donor/recipient chimerism is of major significance in detecting and quantifying engraftment or graft rejection of the donor-derived haematopoietic cells after transplantation. Radioactive analyses of polymorphic microsatellite markers are commonly used for chimerism analyses. We describe an improved, non-isotopic method that is based on the analysis of microsatellite markers in donor and recipient cells using capillary electrophoresis and fluorescence detection. Artificial mixtures of donor and recipient DNA that were generated from peripheral blood mononuclear cells from dog leukocyte antigen-identical siblings were used to analyse the sensitivity of the assay. DNA from dogs that had received HSCT were also analysed in order to demonstrate the feasibility of the method in vivo. For chimerism analyses, six different microsatellite loci were systematically amplified using fluorescent PCR primer. The fluorescent polymerase chain reaction products were separated by capillary electrophoresis using POP4 on a 310 ABI Prism Genetic Analyzer. After electrophoresis, fluorescence signals were automatically sized and quantified using GeneScan software. The method described provides an accurate assessment of haematopoietic chimerism in the canine model with significantly reduced hands-on time compared to conventional gel electrophoresis.
Similar content being viewed by others
REFERENCES
Acquaviva, C., Duval, M., Mirebeau, D., Bertin, R. and Cave, H., 2003. Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Paris-Robert Debre experience. Leukemia,17, 241–246
Buse, E.L., Putinier, J.C., Hong, M.M., Yap, A.E. and Hartmann, J.M., 2003. Performance evaluation of two multiplexes used in short tandem repeat DNA analysis. Journal of Forensic Sciences, 48 , 348–357
Francisco, L.V., Langston, A.A., Mellersh, C.S., Neal, C.L. and Ostrander, E.A., 1996. A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mammalian Genome, 7 , 359–362
Hancock, J.P., Goulden, N.,J., Oakhill, A. and Steward, C.G., 2003. Quantitative analysis of chimerism after allogenic bone marrow transplantation using immunomagnetic selection and fluorescent microsatellite PCR. Leukemia 17, 247–251
Karnes, H.T., Shiu, G. and Shah, V.P., 1991. Validation of bioanalytical methods. Pharmaceutical Research, 8. 421–426
Junghanss, C., Takatu, A., Little, M.T., Maciej Zaucha, J., Zellmer, E., Yunusov, M., Sale, G., Georges, G.E. and Storb, R., 2003. Adoptive immuntherapy against kidney targets in dog-leucocyte antigenidentical mixed hematopoietic canine chimeras. Transplantation 75, 268–274
Ostrander, E.A., Spraque, G. and Rine, J., 1993. Identification and characterization of dinucleotide repeat (CA), markers for genetic mapping in dog. Genomics 16, 207–213
Schraml, E. and Lion, T., 2003, Interference of dye-associated fluorescence signals with quantitative analysis of chimerism by capillary electrophoresis. Leukemia 17, 221–223
Sreenan, J.J., Pettay, J.D., Tbakhi, A., Totos, G., Sandhaus, L.M., Miller, M.L., Bolwell, B. and Tubbs, R.R., 1997. The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting. American Journal of Clinical Pathology 107, 292–298
Storb, R., Yu, C., Wagner, J.L., Deeg, H.J., Nash, R.A., Kiem, H.P., Leisenring, W. and Shulman, H., 1997 Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood, 89 , 3048–3054
Wagner, J.L. and Storb, R., 1996. Preclinical large animal models for hematopoietic stem cell transplantation. Current Opinion in Hematology, 3, 410–415
Wagner, J.L., Burnett, R.C., DeRose, S.A., Francisco, L.V., Storb, R. and Ostrander, E.A., 1996. Histocompatibility testing of dog families with highly polymorphic microsatellite markers. Transplantation, 62, 876–877
Walsh, P.S., Fildes, N.J. and Reynolds, R., 1996, Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Research, 24, 2807–2812
Yu, C., Ostrander, E., Bryant, E., Burnett, R. and Storb, R., 1994. Use of (CA), polymorphisms to determine the origin of blood cells after canine marrow grafting. Transplantation, 58, 701–716
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hilgendorf, I., Weirich, V., Zeng, L. et al. Canine Haematopoietic Chimerism Analyses by Semiquantitative Fluorescence Detection of Variable Number of Tandem Repeat Polymorphism. Vet Res Commun 29, 103–110 (2005). https://doi.org/10.1023/B:VERC.0000047486.01458.c5
Issue Date:
DOI: https://doi.org/10.1023/B:VERC.0000047486.01458.c5