Figure 1. Identification of a GH promoter DMR using mouse BAC transgenes.

A. A mouse BAC transgene encompassing GH gene was modified by homologous recombination to replace the mouse GH gene with the rat GH proximal promoter and the coding sequence for RFP. A second recombination event using the same BAC transgene was used to delete a putative distal LCR (ΔLCR- GH:RFP). Imaging of dissected mouse pituitary, illustrates RFP expression from the WT-GH:RFP but not ΔLCR-GH:RFP transgenic mice. B. WT-GH:RFP protein mirrored endogenous GH expression. Pituitaries were fixed and embedded in paraffin, the tissue sectioned and subjected to indirect immunofluorescence with anti-GH, anti-Prl and anti-TSHβ antibodies using Cy2-cojugated secondary antibodies. The images were merged with images of RFP expression. Note: RFP was nuclear while the hormones were localized to the cytoplasm. C. Pairwise analysis of the rat GH promoter methylation levels by bisulfite sequencing of pituitary DNA from WT-GH:RFP and ΔLCR-GH:RFP transgenic mice. The number indicates the relative position of the CpG from the transcriptional start site. The n-values indicate the number of clones sequenced from transgenic mice and used for the pairwise statistical analysis.