Full-length and internally deleted forms of interleukin-7 are present in horse (Equus caballus) lymph node tissue.
Abstract: Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events. Consequently, the shorter open-reading frames were re-named splice variant (SV) 1 (477bp), 2 (396bp) and 3 (264bp). Organization of the horse IL-7 is predicted to be similar to that in humans with exon 5 deleted from SV1, exons 3, 5 deleted from SV2 and exons 3, 4, and 5 missing from SV3. Each of these open-reading frames has the potential to be stably expressed as demonstrated using a polyclonal antiserum against human IL-7 to visualize the protein products produced when the FL HIL-7 and each SV were molecularly cloned into pCI and transfected in brefeldin A treated HEK 293 cells. Furthermore, addition of supernatants to horse PBMC from HEK cells transfected (without brefeldin A treatment) with pCI HIL-7 FL, pCI HIL-7SV1, pCI HIL-7SV2 and pCI IL-7SV3 all induced significant incorporation of (3)H-thymidine in the presence of sub-stimulatory amounts of concanavalin A compared to supernatants from mock-transfected cells. Therefore, all isoforms of horse IL-7 described in this report have the ability to stimulate proliferative responses in ex vivo horse PBMC cultures.
Publication Date: 2008-05-14 PubMed ID: 18573542DOI: 10.1016/j.vetimm.2008.05.006Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research discusses the discovery of full-length and shorter forms of horse interleukin-7 (a type of protein that regulates immune system responses) from adult horse lymph nodes and the various responses it stimulates in a specific type of cell culture.
About the Research Objectives
- The research aimed at isolating horse IL-7 cDNA (a form of DNA synthesized from a messenger RNA template) from adult lymph node tissues using reverse transcription polymerase chain reaction (RT-PCR) – a laboratory technique used for creating copies of a specific part of the DNA.
- Along with the full-length (FL) frame of 531 base pairs long that encoded 176 amino acids, shorter sequences of 477, 396, and 264 base pairs were also identified.
Findings from the Nucleotide Sequence Analysis
- The analysis showed homology between the full-length sequence and shorter sequences except for the fact that the latter were missing certain internal sequences. This difference was considered consistent with multiple RNA splicing events – a process that removes non-coding regions from the RNA molecule to create mature RNA.
- Based on these findings, the shorter sequences were named as splice variants (SV) 1 (477 base pairs), 2 (396 base pairs), and 3 (264 base pairs).
Predicted Organization of Horse IL-7
- The sequence organization of horse IL-7 is hypothesized to be similar to that in humans. The researchers observed that SV1 lacks exon 5, SV2 lacks exons 3 and 5, and SV3 lacks exons 3, 4, and 5.
- Exons are distinct parts of a gene that contain the actual coding sequence for protein synthesis.
Protein Production and Stimulation of Cell Proliferation
- It was demonstrated that each of the open-reading frames (segments of the DNA that can potentially be translated into protein) has the potential to be stably expressed. This was tested by visualizing the protein products produced when the FL HIL-7 and each splice variant were cloned into a type of vector (pCI) and transferred into a certain type of cells (HEK 293 cells).
- Further, it was shown that the protein isoforms of horse IL-7 could stimulate proliferation in horse PBMC (peripheral blood mononuclear cell) cultures when added to the culture medium. PBMC cultures are commonly used for a variety of immunology research applications.
Cite This Article
APA
Cook RF, Cook SJ, Even DL, Schaffer C, Issel CJ.
(2008).
Full-length and internally deleted forms of interleukin-7 are present in horse (Equus caballus) lymph node tissue.
Vet Immunol Immunopathol, 125(1-2), 126-134.
https://doi.org/10.1016/j.vetimm.2008.05.006 Publication
Researcher Affiliations
- Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA. rfcook1@uky.edu
MeSH Terms
- Amino Acid Sequence
- Animals
- Cell Proliferation / drug effects
- Cloning, Molecular
- DNA, Complementary / genetics
- Horses / genetics
- Horses / immunology
- Interleukin-7 / biosynthesis
- Interleukin-7 / genetics
- Interleukin-7 / immunology
- Leukocytes, Mononuclear / immunology
- Lymph Nodes / immunology
- Molecular Sequence Data
- Protein Isoforms
- RNA / chemistry
- RNA / genetics
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Sequence Alignment
- Transfection / veterinary
Citations
This article has been cited 2 times.- Yu D, Xu L, Liu XH, Fan Y, Lü LB, Yao YG. Diverse interleukin-7 mRNA transcripts in Chinese tree shrew (Tupaia belangeri chinensis). PLoS One 2014;9(6):e99859.
- Siewe BT, Kalis SL, Esteves PJ, Zhou T, Knight KL. A novel functional rabbit IL-7 isoform. Dev Comp Immunol 2010 Aug;34(8):828-36.
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