DRB genotyping in cynomolgus monkeys from China using polymerase chain reaction -sequence-specific primers.
Cynomolgus macaques are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of the macaque major histocompatibility complex (MHC). Because of high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for cynomolgus monkey MHC class II. We developed a simple and rapid polymerase chain reaction-sequence specific primer (PCR-SSP) strategy for Chinese cynomolgus monkey DRB locus typing. Forty Chinese cynomolgus monkeys originating from the Guangxi Province in China were included in the study. Twenty nine cynomolgus monkey allele-specific primer pairs were designed based on published Macaca fascicularis (Mafa)-DRB locus gene sequences. Allele-specific PCR products ranged in size from 143 to 253 bp; 5' and 3' Mafa-DRB locus allele specific primers were located in the second exon. Specific PCR product gel purification was followed by direct sequencing in both directions. Our data showed prominent variability in the number of Mafa-DRB sequences ranging from 2 to 7 per animal. This analysis demonstrated that most of the amplicons were identical to Mafa-DRB sequences that our PCR primers were to amplify. However, 98 to 99% similarity was noticed in the case of Mafa-DRB4*0101, Mafa-DRB*W2101, and Mafa-DRB*W4901 sequences. The observed mismatches were located in nonpolymorphic regions. Thus, haplotype analysis confirmed the existence of allelic associations published earlier. In addition, we propose a new DRB sequence. The established medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in Chinese cynomolgus monkeys.
Wei
Huafeng
H
International Joint Cancer Institute, The Second Military Medical University, Shanghai, People's Republic of China.
Wang
Hao
H
Hou
Sheng
S
Hu
Shuiqing
S
Fan
Kexing
K
Fan
Xiaoqiang
X
Zhu
Tongyu
T
Guo
Yajun
Y
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D005091:Exons; D005838:Genotype; D006684:HLA-DR Antigens / Q000235:genetics*; D059805:HLA-DR alpha-Chains; D008252:Macaca fascicularis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D016415:Sequence Alignment; D013045:Species Specificity
D017931:DNA Primers; D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains