A system for in vivo ablation of MeCP2-deficient neurons in the Rett Syndrome mouse model

The contents of this file relate to the data generated during the PhD research (Biological Sciences) of Katie Paton, under the supervision of Professor Adrian Bird and Professor Susan Rosser, over the period between September 2020 and December 2021. All data within this file were generated during the aforementioned period, and mainly (but not exclusively) for the purposes of the PhD research and production of the thesis. The data are contained within the main folder “PhD data_Katie Paton”. Within this folder 3 folders can be found: 1) Chapter 3_Mecp2∆DTR-Stop line_generation and characterization; 2) Chapter 4+6_Mecp2∆DTR-FLEx line_generation and characterization; 3) Chapter 5_Transgenic Cre driver_charactisation, each containing the data related to the thesis chapter stated in the name. Within each of the three main folders the data is further categorized into sub-folders, each of which are titled with a brief description of the type of experiment the data addresses. Data files are: images (such as .png .jpg .tif), GraphPad datasets (.pzfx), Excel datasets (.xlsx), DNA sequences (.sbd .dna), flow cytometry data (original .fcs or FlowJo-processed .wsp), imaging files made using specialized software (Zeiss .czi, Leica .lif), Typhoon FLA 7000 phosphoimager data (.gel), Incucyte images and analysis data (.jpg .txt) and others. Further information detailing what data is in each of the three main folders and how it relates to the PhD thesis is provided in .txt files. The PhD thesis is provided as a PDF file for reference and guidance. For any questions, please contact Katie Paton or Adrian Bird.

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Rett syndrome (RTT) is an X-linked neurological disorder caused by loss-of-function mutations in the MECP2 gene. Heterozygous RTT females express a phenotype due to mosaic expression of their wild type (WT) copy of MECP2 caused by random X-chromosome inactivation. Furthermore, neurons expressing WT MECP2, which have the capacity to function normally, are interspersed with neurons expressing the mutant allele, which function inappropriately and compromise the whole neuronal network. Re-expression of MeCP2 in Mecp2-mutant adult mice reverses the neurological phenotype. Thus, therapeutic avenues explored so far to treat RTT, focus on repairing MeCP2 expression in MeCP2-deficient neurons. One of the most challenging aspects of this approach is maintaining MeCP2 levels within physiological limits, as both too much or too little MeCP2 expression lead to neurological disease. An alternative approach based on silencing or removal of the functionally mutant neurons from the mosaic neuronal network has not yet been explored, but could in theory be curative. I seek to selectively ablate MeCP2-deficient neurons at different stages in development in the heterozygous mouse model of RTT to determine whether the removal of 50% of neurons in the developing brain is compatible with viability at various developmental stages. The ultimate goal is to ask: does the removal of the MeCP2-deficient neurons ameliorate the RTT-like phenotype?
This thesis describes the development of an inducible in vivo cell ablation system for selective removal of Mecp2 knock-out neurons in the heterozygous RTT mouse model. The Mecp2∆DTRFLEx genetically engineered mouse line was generated which facilitates Cre-dependent expression of the Diphtheria Toxin Receptor (DTR) from the Mecp2 locus in place of the Mecp2 gene. Mice are resistant to Diphtheria toxin (DT), so expression of the DTR in a cell-type of interest, allows ablation of those cells upon DT administration. Un-recombined Mecp2∆DTRFLEx OFF (cre-negative) mice were resistant to high doses of DT and therefore comparable to WT littermates. DTR expression was shown to be activated only in the presence of Cre recombinase. The combination of the Mecp2∆DTRFLEx allele with a transgenic Cre driver allows targeting of DTR expression to Mecp2– cells in the tissue of interest. Preliminary in vivo experiments were carried out to explore the ablation of MeCP2-deficient neurons in neonatal development. This thesis discusses the associated challenges and future applications of this system.

Paton KM; Selfridge J; Guy J; Bird A. A system for in vivo ablation of MeCP2-deficient neurons in the Rett Syndrome mouse model – Katie Paton. Edinburgh DataVault (2021).