Duplication of Stat5-binding sites within the beta-lactoglobulin promoter compromises transcription in vivo.

Achieving high and reliable levels of expression is essential for biotechnological application of transgene technology. In an attempt to maximise transgene expression, we analysed the effect of adding extra Stat5-binding sites, while maintaining their spatial arrangement, to the beta-lactoglobulin promoter. A 195 bp-fragment comprising the three Stat5-binding sites in the beta-lactoglobulin promoter was duplicated directly upstream, 88 bp or 177 bp upstream from the original site. We assessed the effect that addition of extra Stat5-binding sites had in transcriptional activation and chromatin structuring of the promoter, both in vitro in HC11 cells and in vivo in transgenic animals. Duplication of Stat5-binding sites in the beta-lactoglobulin promoter did favour chromatin remodelling in vivo but unexpectedly did not enhance transcriptional activation of the transgene, despite the higher basal and induced expression levels achieved in vitro in HC11 cells. Furthermore, the well-described positive relationship between beta-lactoglobulin transgene copy number and mammary expression was lost in the new lines. These results indicate that chromatin remodelling upon Stat5 binding is not sufficient to activate transgene expression, denote that strategies to achieve transgenic transcriptional activation are not obvious.