Abstract
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 experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). 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. 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 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. 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.
Highlights
Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection
The minigene consists of the endogenous Tdg promoter and terminator sequences flanking the Tdg coding sequence (CDS)
It includes the rabbit β-globin intron at the authentic position of the first intron of the Tdg transcript variant 2 (Genebank NM_172552.4). This intron allows for the expression of the two naturally occurring Tdg splice variants (Um et al, 1998)
Summary
Inducible TDG knockout models to study epigenetic regulation [version 2; peer review: 3 approved]. Schwarz 1, Eliane Grundbacher[1], Alexandra M. Hrovat 1, Jianming Xu1, Anna Kuśnierczyk[2], Cathrine B. Vågbø[2], Primo Schär 1, David Schuermann 1.
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