Inducible TDG knockout models to study epigenetic regulation

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.