Fig. 3

Verification of the proximal enhancer of the chicken NANOG (cNANOG gene) in chicken primordial germ cells (PGCs). a-b Schematic diagram of the constructed cNANOG promoter vectors and luciferase activity in PGCs (a) and DF-1 cells (b). c Prediction of transcription factor (TF)-binding sites in the cNANOG promoter region located from − 250 to + 70 bp. d Multiple alignment of the putative cNANOG proximal enhancer with transcriptional regulatory elements of NANOG genes from mouse, rat, human, cattle, sheep, pig, and chicken. Prediction of mostly conserved POU5F3- and SOX2-binding sites in chicken. e Mutation analysis of putative POU5F3- and SOX2-binding sites in PGCs. f Luciferase activity of the −130/+ 70 bp cNANOG promoter fragment compared with that of mutated promoter constructs. Significant differences are indicated as ** P < 0.01 and *** P < 0.001. Error bar represent the SEs for five replicate reactions