Following the activity of dioxygenases of the ten-eleven-translocation (TET) family that iteratively oxidize 5-methylcytosine (5-mC) to formyl- and carboxylcytosine (5-fC, 5-caC), TDG-induced BER provides a mechanism of active DNA demethylation and, thereby, impact gene regulation ( Cortázar et al., 2011 Cortellino et al., 2011 He et al., 2011 Ito et al., 2011 Schuermann et al., 2016 Shen et al., 2013 Weber et al., 2016). One of those mechanisms includes the thymine DNA-glycosylase (TDG) and the downstream factors of DNA base excision repair (BER), which were shown to be involved in the dynamic, locus-specific regulation of DNA methylation. The underlying mechanisms to ensure this necessary epigenetic plasticity are therefore active and highly dynamic in embryonic stem cells (ESC). To allow for the differentiation to a multitude of cell types during the development of multicellular organisms, stem cells need to respond to a variety of extrinsic and intrinsic developmental cues and integrate these into epigenetically stabilized gene expression patterns during lineage specification. Thus, we provide a tested and well-controlled 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. ![]() The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. We describe the functionality of the engineered miniTdg 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 loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection.
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