Fig. 2

Identification of CVH promoter regions through 5′ and 3′ fragmentation assays. a Schematic diagram of six fragmented constructs of the CVH promoter. Constructs were designed for expression analysis using an eGFP expression vector: 5′ and 3′ random deletion assays were conducted for a 591-bp fragment (−316/+275) and a 410-bp fragment (−135/+275), respectively. For 3′ deletion for a 410-bp fragment (−135/+275), two constructs for a 357-bp fragment (−135/+222) and a 297-bp fragment (−135/+162) contained part of the intron region. b Expression analysis of eGFP under different fragments of the CVH promoter in cultured chicken PGCs. Twenty-four hours after transfection, the expression of eGFP was observed by fluorescence microscopy. Each fragment used for driving eGFP expression was ligated into the NanoLuc luciferase expression vector (pNL1.2-Basic) for measuring promoter activity. The expression of NanoLuc luciferase was measured using Nano-Glo-dual luciferase assays in PGCs (c) and DF-1 (d). Promoter activity was measured as the ratio of NanoLuc luciferase expression levels to internal firefly control and is expressed as relative luciferase units. Scale bar = 100 μm. Different letters (a–e) indicate significant differences (P < 0.05)