Forsøksdyr: TDG-regulated neuroepigenetic mechanisms in the entorhinal-hippocampal spatial memory coding system.

Godkjenningsdato 13.08.2020

Godkjenningsperiode 01.09.2020-31.08.2024

The Thymine-DNA glycosylase (TDG) plays an essential role in the process of DNA demethylation, regulating the epigenetic DNA methylation patterns in the genome. Constitutive knockout of TDG is embryonic lethal. In this project, I will use a Cre-Lox system to knock out TDG in specific brain regions and/or distinct neuronal subtypes. To achieve this, Lox-TDG (background C57Bl/6J) mice will be injected with rAAV-cre viruses in brain regions such as the hippocampus (HPC) and the medial entorhinal cortex (MEC) or crossed with cell-type-specific Cre-mouse lines. I aim to explore TDG-specific neuroepigenetic mechanisms in distinct types of neurons linking to neuronal functions in cognition and behaviour. This project will open a novel research field that links the dynamic DNA demethylation to functionally distinct neurons and neural networks contributing to memory formation.

To minimize the distress of animals and improve animal well-being, I will provide enriched housing conditions, careful handling and caring before, during and after the surgery, and close monitoring during the whole experimental procedure. As for the studies with viral-based TDG knockout, a total number 270 of Lox-TDG mice is planned in histology, behaviour and neuronal recording tasks. Viral-based TDG knockout saves time, space and cost but is only applied locally and may introduce variations due to the quality of different batches of viruses and the experimental procedures. The number of animals could be further reduced by sharing the same mice in experimental approaches for behaviour and neuronal recording studies. As for the cell-type specific TDG-knockout mouse models, Lox-TDG mice will be crossed with cell-type-specific Cre (such as Camk2a-Cre, Grik4-Cre and PV-Cre) mouse lines. For this, 20 of Lox-TDG mice (female) will be used and Cre-mice (male, 2 of each genotype) will be purchased from the Jackson Laboratory (JAX). Although it takes time, space, cost and efforts to generate new mouse models, once the animal models are established and verified, it allows us to explore TDG-specific epigenetic regulation in distinct types of neurons in the whole brain and saves the number of animals for further studies. Camk2a-TDG -/- mice, Grik4-TDG -/- mice, PV-TDG -/- mice and TDG-control mice will be explored in the histology, behaviour and neuronal recording studies. The estimated number of animals will be 60 of each, but the number of animals could be further reduced by sharing the same mice in different approaches. Pilot experiments will be performed first. I will take advantages of the two TDG knockout methodologies and choose the most efficient strategy and use the minimal number of animals to address specific scientific questions.