Inducible TDG knockout models to study epigenetic regulation

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA repair mediated active DNA demethylation, we generated a <em>Tdg </em>minigene model in mice and mouse embryonic stem cells (ESCs). The floxed <em>miniTdg</em> is rapidly and reliably excised by tamoxifen-induced Cre expression in mice and ESCs, depleting TDG to undetectable levels within 24 hours of induction. We describe the functionality of the engineered <em>miniTdg</em> in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any time point in multistep experimental procedures as presented here for neuronal differentiation <em>in vitro</em>. Thus, we provide a thoroughly validate d genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.通过基因敲除或基因编辑手段开展的机制与功能研究，常受到克隆筛选所引发的代偿性细胞适应等混杂效应的干扰。这类问题在研究直接参与遗传或表观遗传维持的因子时尤为突出。为了构建可用于DNA修复介导的主动DNA去甲基化功能与机制研究的遗传工具，我们在小鼠及小鼠胚胎干细胞（ESCs）中构建了<em>Tdg</em>（胸腺嘧啶DNA糖苷酶）迷你基因模型。经loxP位点侧翼标记的<em>miniTdg</em>可在小鼠及ESCs中被他莫昔芬诱导的Cre重组酶表达快速且稳定地切除，诱导后24小时内即可将TDG蛋白耗竭至无法检测的水平。我们详述了工程化构建的<em>miniTdg</em>在小鼠及ESCs（TDGiKO ESCs）中的功能表现，并验证了TDGiKO ESCs的多能性与分化潜能，同时确证了诱导型TDG耗竭的表型特征。这种可精准调控的快速TDG耗竭手段，可在多步骤实验流程的任意时间点实现精准操作，正如本文针对<em>in vitro</em>神经元分化所展示的案例。综上，我们构建并完成了全面验证的遗传工具，可用于TDG在主动DNA（去）甲基化及/或DNA修复过程中的功能与机制研究，且可最大限度降低代偿效应与克隆筛选带来的干扰。