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United States Department of Agriculture

Agricultural Research Service

Title: Use of Antisense Oligonucleotides As a Functional Genomic Research Tool to Elucidate Role of Chicken Myd88 in Toll-Like Receptor Mediated Innate Immune Responses in Vitro

Authors
item He, Louis
item Genovese, Kenneth
item Kogut, Michael

Submitted to: Chicken Genomics and Development Workshop
Publication Type: Abstract Only
Publication Acceptance Date: May 8, 2005
Publication Date: May 8, 2005
Citation: He, H., Genovese, K.J., Kogut, M.H. 2005. Use of antisense oligonucleotides as a functional genomic research tool to elucidate role of chicken MyD88 in toll-like receptor mediated innate immune responses in vitro [abstract]. Chicken Genomics and Development. p. 28.

Technical Abstract: The family of Toll-like receptors (TLRs) recognizes conserved structures found in a broad range of pathogens and initiates innate immune responses. Myeloid differentiation factor 88 (MyD88) is a TIR (Toll/interleukin (IL)-1) family receptor and plays crucial roles in signaling TLRs mediated innate immune responses to infections. The function of MyD88 in innate immune responses has been studied in human and murine cells. Partial sequences of putative chicken MyD88 (chMyD88) have been identified via in silico analysis from available expressed sequence tag (EST) and genomic sequence database. The full length cDNA sequence encoding chMyD88 was obtained using PCR mediated rapid amplification of cDNA ends (RACE). The cDNA encodes chMyD88 of 299 amino acid (aa) residues which shares 70% identity with human MyD88 (296 aa). Using phosphorothioate antisense oligonucleotides (oligos), we have demonstrated the differential role of chMyD88 in TLR4 and TLR9 mediated activation of the chicken macrophage cell line HD11. Lipopolysacharide (LPS), a TLR4 ligand, and CpG-oligodeoxynucleotide (CpG ODN), a TLR9 ligand, stimulate activation of HD11 cells and induce nitric oxide (NO) production. Preincubation of HD11 cells with an antisense oligo abrogated induction of NO by CpG ODN. No inhibitory effect was observed on CpG ODN-induced NO production when an unrelated control oligo was used. On the contrary, neither the antisense nor the control oligo affected LPS-induced NO synthesis. Our results indicate that TLR4 mediates LPS-induced NO production in HD11 cell via a signaling pathway independent of MyD88; whereas TLR9 mediated immune response of HD11 cells to CpG-ODN is MyD88-dependent. Our results confirm the conserved functions of chMyD88 in TLRs mediated innate immune responses similar with murine and human MyD88. Furthermore, our study demonstrated antisense technology can be used as a functional genomic research tool to elucidate or to confirm functions of newly identified genes.

Last Modified: 10/20/2014
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