Lentivirus-Mediated RNAi Knockdown of Chicken ATF3 and MYD88 genes in the HD-11 Cell Line

Both activating transcription factor 3 (ATF3) and myeloid differentiation primary response gene (MYD88) play important roles in the inflammatory response induced by Salmonella during its infection of chickens. To understand the molecular mechanism of how these two genes regulate host response to Salmonella infection in HD11 cells, a chicken macrophage cell line, a lentivirus-mediated RNAi knockdown approach was employed. Three shRNA sequences were designed and synthesized for each gene. Each of these synthesized shRNAs were verified using the TOPO cloning vector and Sanger sequencing, before being cloned into the lentiviral vector PCG-luc-1309 that already contains the reporter green fluorescent protein (GFP). The PCG-luc-1309 vector containing shRNA insert was then co-transfected into 293T cells with the lentiviral packaging plasmid and envelope plasmid pMD.G using Lipofectamine 2000. Transfection efficiency was assayed via fluorescent imaging of GFP expression. Over 70% of confluent 293T cells showed GFP expression. Lentiviral supernatant collected at 48 and 72 hours post transfection was used to infect the HD11 cell line. Initial viral integration rates of fewer than 5% were selected for using Puromycin. Puromycin selection was used to select for strictly fluorescent infected positive cells, resulting in over 95% fluorescence within two weeks. Total RNA and protein was isolated from transfected and control HD11 cells. These isolates are presently being used to assess RNAi knockdown efficiency of ATF3 and MYD88 using qPCR and Western blots.