Pena Ramona N, Whitelaw C Bruce A
Division of Gene Function and Development, Roslin Institute, Roslin EH25 9PS, Midlothian, Scotland, UK.
Biochimie. 2005 Jun;87(6):523-8. doi: 10.1016/j.biochi.2005.02.006. Epub 2005 Mar 23.
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.
实现高水平且可靠的表达对于转基因技术的生物技术应用至关重要。为了使转基因表达最大化，我们分析了在维持β-乳球蛋白启动子中Stat5结合位点空间排列的同时，添加额外Stat5结合位点的效果。包含β-乳球蛋白启动子中三个Stat5结合位点的195 bp片段在原始位点上游88 bp或177 bp处直接上游进行了重复。我们评估了添加额外Stat5结合位点对启动子转录激活和染色质结构的影响，包括在体外HC11细胞中和在体内转基因动物中。β-乳球蛋白启动子中Stat5结合位点的重复确实有利于体内染色质重塑，但出乎意料的是，尽管在体外HC11细胞中实现了更高的基础表达水平和诱导表达水平，但并未增强转基因的转录激活。此外，在新的品系中，β-乳球蛋白转基因拷贝数与乳腺表达之间众所周知的正相关关系消失了。这些结果表明，Stat5结合后的染色质重塑不足以激活转基因表达，这表明实现转基因转录激活的策略并不明确。
