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

• Published in: Xenotransplantation (Københaven) 2005-03, Vol.12 (2), p.121-126
• Main Authors: Ando, Asako, Ota, Masao, Sada, Masaharu, Katsuyama, Yoshihiko, Goto, Rieko, Shigenari, Atsuko, Kawata, Hisako, Anzai, Tatsuya, Iwanaga, Takahiro, Miyoshi, Yukari, Fujimura, Nobuyuki, Inoko, Hidetoshi
• Format: Article
• Language: English
• Subjects: allele-specific amplification; Alleles; Animals; Base Sequence; DNA Primers - genetics; Female; Genotype; Haplotypes - genetics; Histocompatibility Antigens Class I - genetics; Histocompatibility Antigens Class II - genetics; Male; miniature swine; PCR-RFLP; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; SLA genotype; Swine - genetics; Swine - immunology; swine major histocompatibility complex; Time Factors
• ISSN: 0908-665X; 1399-3089
• DOI: 10.1111/j.1399-3089.2005.00204.x

:  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.