Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

中文翻译：

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通过靶向去甲基化控制表观遗传沉默的Tet启动子驱动的转基因表达的重新激活

转基因在细胞培养物和小鼠中的忠实表达经常受到基因座依赖的表观遗传沉默的挑战。我们调查了小鼠ROSA26基因座内Tet受控表达盒的沉默。我们观察到Tet启动子的明显DNA甲基化，同时在mES细胞以及分化细胞和转基因动物中表达丧失。引人注目的是，ROSA26启动子保持活性且无甲基化，表明该沉默机制特异性影响转基因，但不扩散至宿主的染色体附近。为了重新激活Tet盒，构建了合成的融合蛋白并在沉默的细胞中表达。该蛋白质包括十一个11个易位甲基胞嘧啶双加氧酶1（TET-1）的酶促结构域以及Tet阻遏物DNA结合结构域。合成融合蛋白的表达和强力霉素处理使Tet启动子在ROSA26基因座和另一个基因组位点靶向脱甲基，从而挽救了细胞和转基因小鼠中的转基因表达。因此，可诱导的，可逆的和位点特异性的表观遗传学调制是一种重新激活沉默的转基因表达的有前途的策略，而与整合位点无关。