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Figure 4 | BMC Genomics

Figure 4

From: In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene

Figure 4

GnRH tissue specific enhancer and promoter constructs A) The in silico predicted zebrafish gnrh 3 enhancer region located between -976 and -929, featuring binding sites for Oct-1, CREB and Sp1. Coordinates are negatively numbered with the experimental TSS as +1. B) Schematic overview of all zebrafish constructs tested, with nomenclature by sense primer used. The human RFP construct is not shown. The 91 bp deletion (-960 to -869) in the pΔ(A25)-GFP is denoted by an angle in the thin line. The gray and the white boxes correspond to the enhancer and exon 1, respectively. Arrows describe PCR primers, showing name and distance from the upstream end relative to TSS. Restriction sites at the bottom correspond to the tail of the PCR primers used for amplification of alternative promoter fragments. Transient expression data are indicated with +, - or (+), the latter denoting broader cell specificity. PCR primer sequences: A23 (5'aagcttggagtttgcatgttctccct3'), A24 (5'aagcttcctttcttaaaatattgaattatgat3'), A25 (5'ggatcccttcagggatgccaggtctt3'), A26 (5'ctcgaggctgtgtttgttcaagatgagttct3'), A33 (5'aagcttcagggagaacatgcaaact3') and A42 (5'aagcttggaatcagagaccttcttgct3'). C) The putative enhancer featuring binding sites for Oct-1, CREB and Sp1 is 100 % conserved with respect to presence of Oct-1 and CREB for the promoters tested [22, 29, 36–38]. The distance to TSS (+1), the transcription factor binding site and number of nucleotides between binding sites (space), are shown for each promoter. The location of the predicted enhancer in rat GnRH-I is according to data published [54]. D) Schematic overview of primer binding sites and the orientation of the respective primers. The upper line denotes genomic DNA and the lower mRNA, where both are displayed in 5'-3' direction. Exons are shown as gray boxes and primers as arrows.

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