Allele-specific in situ analysis of microchimerism by fluorescence resonance energy transfer (FRET) in nonhuman...

Rhesus monkeys are relevant models for tolerance induction. Hematopoetic chimerism is believed to be one of these strategies. The purpose of this study was to detect donor class I A locus allele specific mRNA in Rhesus monkey kidney recipient. We report here for the first time the results of frequency resonance energy transfer (FRET) hybridization technology in frozen tissues. Frequency resonance energy transfer hybridization was performed by using two Mamu-A*05 allele specific oligonucleotides: a donor probe labeled with FITC and acceptor probe conjugated to Texas Red. The PCR-SSP microchimerism analysis method produced 0.05% and 0.5% of donor DNA for Mamu-DRB1*1002 and Mamu-DRBw301/3 alleles, respectively. The donor cells were detected in mesenteric and/or inguinal lymph nodes, spleen, and liver, where the signal was the strongest. The results of FRET hybridization demonstrated the identical staining pattern in the recipient frozen tissues to that determined by PCR-SSP. Following FRET hybridization, the sections underwent immunohistochemical analysis, which revealed that donor cells had CD8+ phenotype. We demonstrate here for the first time that FRET in situ hybridization technique can be utilized for microchimerism analysis in frozen tissues. We conclude that using two donor mRNA specific oligonucleotide probes, rather than one, produce higher specificity.