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
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
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DOI：
10.1111/j.1399-3089.2005.00204.x
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发表时间：
2005-03-01
影响因子：
3.9
通讯作者：
Inoko, H
中科院分区：
文献类型：
--
作者：
Ando, A;Ota, M;Inoko, H
背景资料：确定新的SLA单倍型的近交小型猪将是有用的，在异源和异种移植的研究，这可以进行代表SLA单倍型的可变组合。方法：在克劳小型猪，两个单倍型（C1和C2）和一个交叉单倍型（C3）已分配的三个SLA经典的I类基因和两个SLA II类基因的RT-PCR产物的核苷酸序列测定。为筛选小型克劳恩猪SLA Ⅰ、Ⅱ类纯合子，本研究采用聚合酶链反应-序列特异性引物（PCR-SSP）和聚合酶链反应-限制性片段长度多态性（PCR-RFLP）相结合的方法，建立了一种快速、简便的SLA Ⅰ、Ⅱ类DNA分型方法（PCR-RFLP）技术。设计了7对等位基因特异性引物，用于扩增3个SLA I类基因SLA-1、SLA-2和SLA-3以及1个SLA II类基因DRB 1的第二外显子。此外，基于PCR-RFLP模式在SLA-DQB 1基因，两个等位基因变异识别的第二个外显子在克劳小型猪。采用PCR-SSP和PCR-RFLP相结合的方法对5个家系的22个样本进行了c1、c2和c3三种单倍型的简单鉴定。一个单一的等位基因在每个I类和II类基因也观察到7个样本SLA I类和II类纯合子与C1或C2单倍型。结论：结合PCR-SSP和PCR-RFLP方法，有利于快速识别的三个单倍型和SLA I类和II类纯合子在个人克劳小型猪。
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.