Development of a simple <i><scp>SLA</scp>‐1</i> copy‐number‐variation typing and the comparison of typing accuracy between real‐time quantitative and droplet digital <scp>PCR</scp>

Lee, Juyoung; Le, Minh-Tri; Choi, Mi Hyun; Le, Van Chanh Quy; Choi, Hojun; Lee, Hyejeong; Song, Hyuk; Kim, Jin‐Hoi; Park, Chankyu

doi:10.1111/age.12785

Cited by 3 publications

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“…Typing PAMs were performed using genomic DNA isolated using NucleoSpin Blood kit (Macherey-Nagel, Düren, Germany), and SLA-1 , SLA-2 and SLA-3 classical SLA I genes. Typing was performed following previously established protocols [ 35 , 36 , 37 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

et al. 2021

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The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

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Section: Methodsmentioning

confidence: 99%

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

et al. 2021

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Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds

Ahn

Jeon

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et al. 2022

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SLA-1 Genetic Diversity in Pigs: Extensive Analysis of Copy Number Variation, Heterozygosity, Expression, and Breed Specificity

Choi

Lee

et al. 2020

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Swine leukocyte antigens play indispensable roles in immune responses by recognizing a large numberof foreign antigens and thus, their genetic diversity plays a critical role in their functions. in this study, we developed a new high-resolution typing method for pig SLA-1 and successfully typed 307 individuals from diverse genetic backgrounds including 11 pure breeds, 1 cross bred, and 12 cell lines. We identified a total of 52 alleles including 18 novel alleles and 9 SLA-1 duplication haplotypes, including 4 new haplotypes. We observed significant differences in the distribution of SLA-1 alleles among the different pig breeds, including the breed specific alleles. SLA-1 duplication was observed in 33% of the chromosomes and was especially high in the biomedical model breeds such as SNU (100%) and NIH (76%) miniature pigs. Our analysis showed that SLA-1 duplication is associated with the increased level of SLA-1 mRnA expression in porcine cells compared to that of the single copy haplotype. therefore, we provide here the results of the most extensive genetic analysis on pig SLA-1. open Scientific RepoRtS | (2020) 10:743 | https://doi.org/10.1038/s41598-020-57712-5www.nature.com/scientificreports www.nature.com/scientificreports/ SLA-1, SLA-2, and SLA-3 are constitutively expressed classical MHC class I genes, but their expression may vary depending on the genetic differences. For example, SLA-1 is duplicated in the haplotypes Hp-2.0, Hp-8.0, Hp-11.0, Hp-12.0, Hp-19.0, Hp-20.0, and Hp-27.0 14,24-26 . In addition, SLA-1, 3, and 6 were not expressed in the haplotypes Hp-3.0, Hp-2.0, and Hp-5.0, respectively 24 . Recently, a method was reported to estimate the copy number of SLA-1 and to facilitate our understanding on the functional aspect of SLA-1 duplication 27 . The frequency of SLA-1 duplication could be abundant, but the detailed functional analysis is not been available.Therefore, we developed a genomic DNA based high resolution SLA-1 typing method with high accuracy regardless of CNVs and present the extensive analysis results of SLA-1 diversity including new alleles and haplotypes, and allelic distribution among different breeds. We also analyzed the level of SLA-1 expression in pig cells according to their copy numbers which could affect MHC class I-specific immune responses. The information presented in this study should contribute to improving our understanding on the genetic polymorphisms of SLA-1 in diverse pig breeds. ResultsDetermination of the SLA-1 specific region. To develop a genomic DNA-based typing method of SLA-1, the determination of conserved locus specific region to design SLA-1 specific primers is required. We previously reported the locus specific nucleotide sequence variations at the downstream promoter region from six classical SLA class I-related genes including . Here, we extended the results by incorporating genomic sequences from additional cloning and sequence analysis. As a result, we identified a SLA-1 specific motif between the TATA box and the CAP site in the 5ʹ UTR and designe...

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Development of a simple SLA‐1 copy‐number‐variation typing and the comparison of typing accuracy between real‐time quantitative and droplet digital PCR

Cited by 3 publications

References 10 publications

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds

SLA-1 Genetic Diversity in Pigs: Extensive Analysis of Copy Number Variation, Heterozygosity, Expression, and Breed Specificity

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