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Small interfering RNA-mediated knockdown of chicken interferon-γ expression

Research output: Contribution to journalArticle

  • Kamran Haq
  • Sarah K Wootton
  • Neda Barjesteh
  • Michael St Paul
  • Serguei Golovan
  • Andrew J Bendall
  • Shayan Sharif

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Original languageEnglish
Pages (from-to)319-27
Number of pages9
JournalJournal of Interferon and Cytokine Research
Volume33
Issue number6
DOIs
Publication statusPublished - Jun 2013

Abstract

Interferon (IFN)-γ is a cytokine with a variety of functions, including direct antiviral activities and the capacity to polarize T-cells. However, there is limited information available about the function of this cytokine in the avian immune system. To gain a better understanding of the biological relevance of IFN-γ in chicken immunity, gain-of-function (upregulation) and loss-of-function (downregulation) studies need to be conducted. RNA interference (RNAi), a technique employed for downregulating gene expression, is mediated by small interfering RNA (siRNA), which can trigger sequence-specific gene silencing. In this regard, sequence specificity and delivery of siRNA molecules remain critical issues, especially to cells of the immune system. Various direct and indirect approaches have been employed to deliver siRNA, including the use of viral vectors. The objectives of the present study were to determine whether RNAi could effectively downregulate expression of chicken IFN-γ in vitro, and investigate the feasibility of recombinant adeno-associated virus to deliver siRNA in vitro as well. Three 27-mer Dicer substrate RNAs were selected based on the chicken IFN-γ coding sequence and transfected into cells or delivered using a recombinant avian adeno-associated virus (rAAAV) into a chicken fibroblast cell line expressing chIFN-γ. The expression of chIFN-γ transcripts was significantly downregulated when a cocktail containing all three siRNAs was used. Expression of endogenous IFN-γ was also significantly downregulated in primary cells after stimulation with a peptide. Further, significant suppression of IFN-γ transcript was also observed in vitro in cells that were treated with rAAAV, expressing siRNA targeting IFN-γ. Off-target effects in the form of triggering IFN responses by RNAi, including expression of chicken 2',5'-oligoadenylate synthetase and IFN-α, were also examined. Our results suggest that siRNAs selected were effective at downregulating IFN-γ in vitro both when delivered directly as well as when expressed by an rAAAV-based vector.

    Research areas

  • Animals, Cell Line, Chickens, Dependovirus, Down-Regulation, Fibroblasts, Gene Knockdown Techniques, Gene Silencing, Genetic Vectors, HEK293 Cells, Humans, Interferon-gamma, RNA Interference, RNA, Small Interfering

ID: 13158645