Background
The association between chimerism and immunologic tolerance in transplantation has shown a path toward eliminating the need for lifelong immunosuppression We have been investigating vascularized composite allograft (VCA) facial transplantation in a well-established non-human primate (NHP) model and have observed transient chimerism without the use of preconditioning therapies when these grafts include vascularized bone marrow The detection of chimerism in the Mauritian cynomolgus macaque (MCM) model relies on flow cytometry with limited ability to detect microchimerism We explored the use of next generation quantitative sequencing as a method of chimerism detection in our VCA model. Methods: Non-specific PCR primers were designed in a conserved region to amplify all Mafa-B major alleles for each of the 7 haplotypes (M1-M7) in MCMs Two animals DNA were initially used: with M3/M3 and M6/M6 haplotypes After confirming PCR efficiency and that products were of appropriate length the primers were coupled with barcodes for use with the ion torrent sequencing system The barcoded primers were added to mixed DNA from M3 and M6 animals and amplified for 25 cycles These products were purified and then sequenced by ion torrent.
Results
Initial efficiency was determined with the non-barcoded primers on 2 different animals (M3/M3, M6/M6) to ensure our conditions and primers were at an optimal range These haplotypes had efficiencies in a reasonable range between 90–105% The addition of the barcode to these primers resulted in a drop in the PCR efficiencies to 77% and 79% respectively both with R2 values of 099 Despite similar but decreased efficiencies, sequencing was subsequently performed Sequencing results indicated that both haplotypes were present at inexact ratios, thus not permitting quantitative assessments of chimerism.
Conclusions
Real time PCR and next generation quantitative sequencing technologies are feasible techniques to detect chimerism in our MCM model Optimization of PCR reaction conditions for barcoded primers are necessary for quantitative interpretations Furthermore, more specific primers for sets of haplotypes may be necessary given the genetically close nature of MCM's