Mapping Promoter Region of chCH25H

To identify the promoter region of chCH25H, the 2077 bp nucleotide sequences before the CH25H coding region were truncated into 6 segments to construct different dual luciferase reporter vectors. The first nucleotide before the chCH25H coding region is numbered −1, and the different dual luciferase reporter vectors are named PGL3- p2077(−2077/−1), p1000 (−1000/−1), p500 (−500/−1), p250 (−250/−1), p125 (−125/−1) and p75 (−75/−1), respectively (Figure 4A). The primers used to construct PGL3 plasmids were showed in Supplementary Table S1.
The PGL3 plasmids (p2077, p1000, p500, p250, p125, p75) were co-transfected with pRL-TK in DF1 cells, respectively. The pGL3-basic was co-transfected with pRL-TK as a control. Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA). Luminescence was measured using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Shoreline, WA, USA). Firefly luciferase activities were normalized to Renilla luminescence in each well.
Furthermore, the common interferon stimulated response elements (ISRE) consensus 5’ A/GGTTTCN(1–2)TTTCC/T3’ or its reverse complement, and the common gamma activated sequence (GAS) consensus 5’ TTNCNNNAA3’ were screened at upstream of chCH25H according to previous studies [23 (link)].