Chimerism analysis by sex determining region Y (SRY) and major histocompatibility complex markers in non-human primates using quantitative real-time polymerase chain reaction.
The ability to discriminate and further quantify the proportion of donor and host cells is essential in hematopoietic stem cell transplant protocols. In human sex-mismatched transplants, this can be easily accomplished by the use of commercially available fluorescent in situ hybridization (FISH) probes. In many animal models, including non-human primates, this methodology is not possible due to the lack of commercially available FISH probes. In animal models, donor cell detection could be accomplished if there is a known species-specific sex determining region Y (SRY) (male) or other unique DNA sequence using either semiquantitative or quantitative real-time polymerase chain reaction (PCR). The use of real-time quantitative PCR has the obvious advantage of providing detailed enumeration of the percentage of donor cells present. We report the development of extremely sensitive primer and probe combinations for male (SRY) and major histocompatibility complex (MHC)-DQA sequences in the macaque and baboon non-human primate models. This assay has a sensitivity of a five-log range and can detect less than four target cells in the presence of 10(5) background cells (approximately 0.001%) and fetal DNA obtained from maternal serum from Macaca nemestrina. The SRY (male) primer and probe combination has similar sensitivity in Macaca fasicularis, Macaca mulatta, and Papio cynocephalus anubis.
Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
A J
AJ
Sieverkropp
R G
RG
Andrews
R A
RA
Andrews
L
L
Gaur
L E
LE
Shields
D000818:Animals; D046528:Chimerism; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D005821:Genetic Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008251:Macaca; D008285:Major Histocompatibility Complex / Q000235:genetics*; D008297:Male; D010215:Papio; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014998:Y Chromosome
D017931:DNA Primers; D004247:DNA