Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR-SSP and PCR-RFLP methods

Abstract

Background: Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. Methods: In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Results: Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. Conclusions: The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine. © Blackwell Munksgaard, 2005.