Publication
Title
An adapted GeneSwitch toolkit for comparable cellular and animal models : a proof of concept in modeling Charcot-Marie-Tooth neuropathy
Author
Abstract
Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch (TM) technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch (TM) cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.
Language
English
Source (journal)
International journal of molecular sciences
Publication
2023
ISSN
1422-0067
1661-6596
DOI
10.3390/IJMS242216138
Volume/pages
24 :22 (2023) , p. 1-18
Article Reference
16138
ISI
001114555500001
Pubmed ID
38003325
Full text (Publisher's DOI)
Full text (open access)
UAntwerpen
Faculty/Department
Research group
Project info
Exploring the role of phosphor-signaling in the etiology of tRNA- synthetase-associated peripheral neuropathies.
Exploring the tissue-specific disease mechanisms underlying tRNA-synthetase-associated peripheral neuropathies.
Publication type
Subject
Affiliation
Publications with a UAntwerp address
External links
Web of Science
Record
Identifier
Creation 09.01.2024
Last edited 11.01.2024
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