Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.
Abstract: Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array, and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high-resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population.
Publication Date: 2010-08-04 PubMed ID: 20683590PubMed Central: PMC3359745DOI: 10.1007/s00251-010-0463-yGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
- Arabian Horses
- Biotechnology
- Cloning
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- DNA
- Equine Diseases
- Equine Health
- Equine Science
- Genetics
- Horse Breeds
- Immunology
- In Vitro Research
- Laboratory Methods
- Major Histocompatibility Complex (MHC)
- Microbiology
- Molecular biology
- Reproduction
- Veterinary Research
- Veterinary Science
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research paper is about the development of a DNA microarray that successfully detects major histocompatibility complex (MHC) class I sequences (part of the immune system) in horses, which can aid in studying T lymphocyte immune responses.
Research Purpose and Method
- The main aim of the research was to create an accurate and efficient molecular-based typing method for equine MHC class I, which would improve the study of T lymphocyte immune responses in horses.
- The authors decided to use a DNA microarray as it is a high-throughput technology that allows for the detection of different sequences at a time, thus enabling an efficient approach to identifying MHC class I.
- The study started with cloning and sequencing of RT-PCR products to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses from a closed Arabian horse breeding herd.
- After the identification phase, sequence-unique, conserved and random nucleotide oligonucleotide microarray probes were designed and spotted onto an epoxy-coated mask slide. This set up allows for precise detection of individual sequences.
Results and Conclusions
- The microarray was successful in consistently detecting eight out of nine classical MHC class I transcripts, thus proving its efficiency over regular cloning and sequencing methods.
- Further, the researchers were able to make ELA haplotypic associations using the detected MHC class I sequences.
- The microarray was then tested on a group of ELA disparate horses and ponies, where it was able to find six new sequences that were previously undetected, further proving the efficiency and specificity of the array method over serology and cloning and sequencing.
- The authors concluded that the microarray method is more efficient at detecting expressed classical MHC class I sequences in this defined population of horses than traditional methodologies.
- Moreover, they proposed that the microarray could be made useful in a broader population with the additions of more probes.
- The overall contribution of this study lies in its development of a high-resolution MHC class I typing method for horse immune studies which can be deployed in a broader horse population in future.
Cite This Article
APA
Ramsay JD, Leib SR, Orfe L, Call DR, Tallmadge RL, Fraser DG, Mealey RH.
(2010).
Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.
Immunogenetics, 62(9), 633-639.
https://doi.org/10.1007/s00251-010-0463-y Publication
Researcher Affiliations
- Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA.
MeSH Terms
- Animals
- Biomarkers / metabolism
- Gene Expression Profiling
- Genes, MHC Class I / genetics
- Haplotypes / genetics
- Horses / genetics
- Oligonucleotide Array Sequence Analysis
- RNA, Messenger / genetics
- Reverse Transcriptase Polymerase Chain Reaction
Grant Funding
- K02 AI073101 / NIAID NIH HHS
- AI073101 / NIAID NIH HHS
References
This article includes 21 references
- Albright-Fraser DG, Reid R, Gerber V, Bailey E. Polymorphism of DRA among equids.. Immunogenetics 1996;43(5):315-7.
- Bailey E. Identification and genetics of horse lymphocyte alloantigens.. Immunogenetics 1980;11(5):499-506.
- Barbis DP, Maher JK, Stanek J, Klaunberg BA, Antczak DF. Horse cDNA clones encoding two MHC class I genes.. Immunogenetics 1994;40(2):163.
- Beck S, Trowsdale J. The human major histocompatability complex: lessons from the DNA sequence.. Annu Rev Genomics Hum Genet 2000;1:117-37.
- Call DR, Bakko MK, Krug MJ, Roberts MC. Identifying antimicrobial resistance genes with DNA microarrays.. Antimicrob Agents Chemother 2003 Oct;47(10):3290-5.
- Carpenter S, Baker JM, Bacon SJ, Hopman T, Maher J, Ellis SA, Antczak DF. Molecular and functional characterization of genes encoding horse MHC class I antigens.. Immunogenetics 2001 Dec;53(9):802-9.
- Chung C, Leib SR, Fraser DG, Ellis SA, McGuire TC. Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.. Eur J Immunogenet 2003 Dec;30(6):387-96.
- Ellis SA, Bontrop RE, Antczak DF, Ballingall K, Davies CJ, Kaufman J, Kennedy LJ, Robinson J, Smith DM, Stear MJ, Stet RJ, Waller MJ, Walter L, Marsh SG. ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005.. Immunogenetics 2006 Jan;57(12):953-8.
- Ellis SA, Martin AJ, Holmes EC, Morrison WI. At least four MHC class I genes are transcribed in the horse: phylogenetic analysis suggests an unusual evolutionary history for the MHC in this species.. Eur J Immunogenet 1995 Jun;22(3):249-60.
- Elsner HA, Blasczyk R. Immunogenetics of HLA null alleles: implications for blood stem cell transplantation.. Tissue Antigens 2004 Dec;64(6):687-95.
- Fraser DG, Bailey E. Polymorphism and multiple loci for the horse DQA gene.. Immunogenetics 1998 May;47(6):487-90.
- Hurley CK, Wade JA, Oudshoorn M, Middleton D, Kukuruga D, Navarrete C, Christiansen F, Hegland J, Ren EC, Andersen I, Cleaver SA, Brautbar C, Raffoux C. A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors.. Tissue Antigens 1999 Apr;53(4 Pt 1):394-406.
- Lazary S, Antczak DF, Bailey E, Bell TK, Bernoco D, Byrns G, McClure JJ. Joint Report of the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, Baton Rouge, Louisiana, 31 October-1 November 1987.. Anim Genet 1988;19(4):447-56.
- Letowski J, Brousseau R, Masson L. Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.. J Microbiol Methods 2004 May;57(2):269-78.
- McGuire TC, Leib SR, Mealey RH, Fraser DG, Prieur DJ. Presentation and binding affinity of equine infectious anemia virus CTL envelope and matrix protein epitopes by an expressed equine classical MHC class I molecule.. J Immunol 2003 Aug 15;171(4):1984-93.
- Mealey RH, Lee JH, Leib SR, Littke MH, McGuire TC. A single amino acid difference within the alpha-2 domain of two naturally occurring equine MHC class I molecules alters the recognition of Gag and Rev epitopes by equine infectious anemia virus-specific CTL.. J Immunol 2006 Nov 15;177(10):7377-90.
- Tallmadge RL, Campbell JA, Miller DC, Antczak DF. Analysis of MHC class I genes across horse MHC haplotypes.. Immunogenetics 2010 Mar;62(3):159-72.
- Tallmadge RL, Lear TL, Antczak DF. Genomic characterization of MHC class I genes of the horse.. Immunogenetics 2005 Nov;57(10):763-74.
- Terasaki PI, Bernoco D, Park MS, Ozturk G, Iwaki Y. Microdroplet testing for HLA-A, -B, -C, and -D antigens. The Phillip Levine Award Lecture.. Am J Clin Pathol 1978 Feb;69(2):103-20.
- Warsen AE, Krug MJ, LaFrentz S, Stanek DR, Loge FJ, Call DR. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.. Appl Environ Microbiol 2004 Jul;70(7):4216-21.
- Williams TM. Human leukocyte antigen gene polymorphism and the histocompatibility laboratory.. J Mol Diagn 2001 Aug;3(3):98-104.
Citations
This article has been cited 2 times.- Vasoya D, Tzelos T, Benedictus L, Karagianni AE, Pirie S, Marr C, Oddsdóttir C, Fintl C, Connelley T. High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure.. Genes (Basel) 2023 Jul 10;14(7).
- Sadeghi R, Moradi-Shahrbabak M, Miraei Ashtiani SR, Miller DC, Antczak DF. MHC haplotype diversity in Persian Arabian horses determined using polymorphic microsatellites.. Immunogenetics 2018 May;70(5):305-315.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
