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In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

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Inducible TDG knockout models to study epigenetic regulation.

Simon D Schwarz¹, Eliane Grundbacher¹, Alexandra M Hrovat¹

¹Department of Biomedicine, University of Basel, Basel, 4058, Switzerland.

|November 4, 2020

Summary

Researchers developed a new genetic tool using a minigene of thymine-DNA glycosylase (TDG) in mice and stem cells. This tool enables rapid and controlled depletion of TDG for studying DNA repair and demethylation with minimal confounding effects.

Keywords:

Active DNA Demethylation Base Excision Repair Cre/loxP Embryonic Stem Cells Minigene Neuronal Differentiation TDG Tamoxifen

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Area of Science:

Molecular Biology
Epigenetics
Genetics

Background:

Gene disruption studies can be confounded by compensatory cellular adaptations.
Investigating DNA repair and epigenetic maintenance factors requires precise genetic tools.
Thymine-DNA glycosylase (TDG) plays a role in DNA repair and active DNA demethylation.

Purpose of the Study:

To generate a controllable genetic tool for investigating thymine-DNA glycosylase (TDG) function.
To enable mechanistic studies of DNA repair-mediated active DNA demethylation.
To minimize confounding effects from clonal selection and adaptive responses.

Main Methods:

Generation of mouse and murine embryonic stem cell (ESC) models with a floxed minigene of TDG (miniTdg).
Tamoxifen-inducible Cre-mediated excision of the miniTdg to achieve rapid and complete TDG depletion.
Validation of TDG depletion efficiency, pluripotency, and differentiation potential of engineered ESCs (TDGiKO ESCs).

Main Results:

Engineered TDGiKO mice and ESCs demonstrate rapid and reliable depletion of TDG upon tamoxifen induction.
TDG depletion was confirmed to be effective, reaching undetectable levels within 24 hours.
TDGiKO ESCs maintain pluripotency and differentiation potential, allowing for controlled experimental manipulation.

Conclusions:

A novel, well-controlled genetic tool (TDGiKO) has been developed for studying TDG.
This tool facilitates precise temporal investigation of TDG's role in DNA (de)methylation and repair.
The system minimizes adaptive effects, providing a more accurate platform for mechanistic studies.