Inducible Overexpression and Targeted Genome Editing of Chicken IRF7 in DF-1 Cell Line

Little is known about the function of chicken interferon regulatory factor (IRF7) while the major function of IRF7 in mammals is regulating type I interferons (IFNs). Our previous study revealed a link between chicken IRF7 and the regulation of Rig-I-like receptor and Toll-like receptor signaling pathways in response to dsRNA. In order to better understand underlying mechanism of how chicken IRF7 regulates the antiviral response against avian influenza virus (AIV), IRF7 conditional over-expression and knock-out DF-1 cell models were further developed. Cumate inducible over-expression system was chosen to avoid the antibiotics (eg. Tetracycline for Tet-on & off system) exposure to the cells during the in vitro pathogen infection. The expression level of IRF7 was linearly induced as increasing amounts of cumate were added and IFNB expression had significant positive correlation with the IRF7 induction. Multiplex genome editing approach was applied using CRISPR/Cas system to mediate targeted genomic deletion. Two spacers that are targeting near the start and stop codon sites of IRF7 were designed to induce concurrent double-stranded breaks followed by nonhomologous end joining to delete the complete IRF7 CDS on the genome (~3kb). The results revealed that 4.5% (4/88) of alleles had large targeted genomic deletion that does not have the IRF7 CDS in the clonal DF-1 colonies. Further in vitro AIV infection studies are followed by functional genomics approaches using these modified cell lines to elucidate the molecular mechanism of avian host immune response against AIV.