Retinoic acid inducible gene I (RIG-I) is an innate immune RNA sensor which can detect viral infection such as influenza viruses. Duck but not chicken has an RIG-I gene. However, the immune responses could be induced in chicken cells by transferring the duck RIG-I transgene. However, effects of other pathogen-recognition receptor (PRR) genes such as Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5) could not be ruled out. In this study, we knocked out TLR3 and MDA5 genes using gene-editing protocol, and stably transferred the duck RIG-I transgene into TLR3/MDA5 double knockout (KO) chicken DF1 cells. We investigated the antiviral responses induced by duck RIG-I in chicken cells. Duck RIG-I induced the expression of interferon-stimulated genes (ISGs) and inflammatory cytokines such as interferon regulatory factor 7 (IRF7), interferon β (IFNβ), Mx1, and protein kinase R1 (PKR1) after treatment with polyinosinic: polycytidylic acid (poly I:C) in TLR3/MDA5 double KO DF1 cells. Additionally, to examine the duck RIG-I signaling cascade, we knocked out mitochondrial antiviral-signaling protein (MAVS), which encodes an antiviral signaling factor in innate immunity. Duck RIG-I in TLR3/MDA5/MAVS triple KO DF1 cells did not activate downstream expression of ISGs. Finally, to analyze the global signaling pathways of duck RIG-I in chicken cells, next-generation sequencing of total mRNAs with and without poly I:C treatment was conducted. In conclusion, duck RIG-I mediated antiviral signaling independently of TLR3 and MDA5, and MAVS induced and stimulated ISGs by duck RIG-I in chicken cells.

Innate immunity, as an organism's first line of defense, plays a crucial role in rapidly responding to and protecting the body against invading pathogens. As a cytosolic RNA sensor for viral infections, including infections caused by influenza virus, the innate immune system in chickens has 2 major pathogen-recognition receptors (PRRs): Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5). The signaling pathways activated by PRRs are complex, systemic processes that underlie the response to foreign molecules. In this study, we investigated the interactions among MDA5, mitochondrial antiviral signaling protein (MAVS), and stimulator of interferon genes (STING) signaling in chicken cells. To exclude the effects of TLR3, we transfected the clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) expression vector and TLR3-targeted gRNA plasmid into chicken DF-1 cells. We selected TLR3-knockout (KO) cell line and sequentially, we established 2 double-KO cell lines: TLR3-MAVS KO and TLR3-STING KO. After treatment with polyinosinic:polycytidylic acid (poly(I:C)), type I interferon (IFN), IFN-stimulated gene, and antiviral gene (IFN regulatory factor 7, IFNβ, Mx1, and protein kinase R1) expression was not completely activated in TLR3-MAVS KO cells, whereas it was consistently upregulated in wild-type and TLR3-STING KO DF-1 cells. These results suggest that STING is not an intermediator between MDA5 and MAVS; moreover, it does not directly interact with MDA5 during innate immune activation in chicken DF-1 cells.

Objective The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. Methods By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. Results We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of ZGFP-knockin chickens. By mating ZGFP-knockin females (ZGFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (ZGFP/Z) even before hatching. Conclusion Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.