Small Interfering RNA-Mediated Knockdown of Chicken Interferon-γ Expression

Interferon (IFN)-gamma 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-gamma 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-gamma 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-gamma coding sequence and transfected into cells or delivered using a recombinant avian adeno-associated virus (rAAAV) into a chicken fibroblast cell line expressing chIFN-gamma. The expression of chIFN-gamma transcripts was significantly downregulated when a cocktail containing all three siRNAs was used. Expression of endogenous IFN-gamma was also significantly downregulated in primary cells after stimulation with a peptide. Further, significant suppression of IFN-gamma transcript was also observed in vitro in cells that were treated with rAAAV, expressing siRNA targeting IFN-gamma. Off-target effects in the form of triggering IFN responses by RNAi, including expression of chicken 2',5'-oligoadenylate synthetase and IFN-alpha, were also examined. Our results suggest that siRNAs selected were effective at downregulating IFN-gamma in vitro both when delivered directly as well as when expressed by an rAAAV-based vector.