Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification

Major histocompatibility complex (MHC) class II molecules play a vital role in the regulation of T-cell functions in the mammalian immune system. Two key features characterize the polymorphism of MHC haplotypes in humans and non-human primates: the existence of a large number of alleles, and the hig h degree of genetic diversity between those alleles. Rhesus monkeys and Chi mpanzees have been extensively used as relevant models for human diseases a nd transplantation We have investigated DRB genes in 19 macaques, members o f 3 families, using polymerase chain reaction with sequence-specific primer s (PCR-SSP) and denaturing gradient gel electrophoresis (DGGE). After ampli fication PCR products were purified and subjected direct sequencing. Seven animals (Madison #1) were typed by DDGE also. We report that the DRB haplot ypes defined by PCR-SSP exhibit a high degree of concordance with the data obtained by DGGE and direct sequening. Our data show prominent variability in the number of DRB1 alleles ranging from id, per genotype within these fa milies, This analysis demonstrated that most of the amplicons were identica l to Mamu-DRB alleles that our PCR primers were to amplify. However, 98-99% similarity was noticed in the case of Mamu-DRB1*0303, Mamu-DRB6*0103 and M amu-DRB*W201 alleles. The observed mismatches were located in non polymorph ic regions. Thus, family studies in rhesus macaques performed by molecular methods confirmed the multiplicity of Mamu-DRB1 alleles per haplotype and t he existence of allelic associations published earlier. In addition, we pro pose 3 more DRB allele associations (haplotypes): Mamu-DRB1*04-DRB5*03; Mam u-DRB1*04-*DRB*W5; Mamu-DRB1*04*W2. The proposed medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing met hod for detecting polymorphisms of DRB genes in rhesus monkeys.