Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression

Kennosuke Ichikawa; Yuzuha Motoe; Ryo Ezaki; Mei Matsuzaki; Hiroyuki Horiuchi

doi:10.1016/j.bbrep.2021.101084

Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression

Biochem Biophys Rep. 2021 Jul 26:27:101084. doi: 10.1016/j.bbrep.2021.101084. eCollection 2021 Sep.

Authors

Kennosuke Ichikawa¹, Yuzuha Motoe¹, Ryo Ezaki¹, Mei Matsuzaki¹, Hiroyuki Horiuchi¹

Affiliation

¹ Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8528, Japan.

Abstract

Waterfowls, such as ducks, are natural hosts of avian influenza virus (AIV) and can genetically limit the pathogenicity. On the other hand, some AIV strains cause severe pathogenicity in chickens. It is suggested that differences in the pathogenicity of AIV infection between waterfowls and chickens are related to the expression of retinoic acid-inducible gene I (RIG-I), a pattern recognition receptor that chickens evolutionally lack. Here, we knocked-in the duck RIG-I bearing the T2A peptide sequence at the 3' region of the Mx, an interferon-stimulated gene (ISG), in chicken embryo fibroblast cells (DF-1) using the precise integration into target chromosome (PITCh) system to control the duck RIG-I expression in chickens. The expression patterns of the duck RIG-I were then analyzed using qPCR. The knocked-in DF-1 cells expressed RIG-I via the stimulation of IFN-β and poly(I:C) in a dose-dependent manner. Moreover, poly(I:C) stimulation in the knocked-in DF-1 cells upregulated RIG-I-like receptor (RLR) family signaling pathway-related genes IFN-β, OASL, and IRF7. The IFN-β-dependent expression of RIG-I and upregulation of IFN-β in the poly(I:C) stimulation demonstrated a positive-feedback loop via RIG-I, usually evident in ducks. Overall, this novel strategy established RIG-I-dependent immune response in chickens without overexpression of RIG-I and disruption of the host genes.

Keywords: AIV, avian influenza virus; Avian influenza virus; DSB, DNA double-strand break; HPAIV, highly pathogenic avian influenza virus; IFNs, interferons; IRF7, interferon regulatory factor 7; ISG, interferon-stimulated gene; ISRE, IFN-stimulated response element; Innate immune response; Interferons; Knock-in; LPAIV, low pathogenic avian influenza virus; MMEJ, microhomology-mediated end-joining; OASL, 2′-5′-oligoadenylate synthase-like protein; PITCh, precise integration into target chromosome; Precise integration into target chromosome; RIG-I, retinoic acid-inducible gene I; RLR, RIG-I-like receptor; Retinoic acid-inducible gene I.