Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

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dc.contributor.authorCarlos-De Clercq, Nina
dc.contributor.authorBreen, Lisa
dc.contributor.authorMcCarthy, Tommy
dc.date.accessioned2024-10-11T14:31:13Z
dc.date.available2024-10-11T14:31:13Z
dc.date.issued2023
dc.description.abstractBackground: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.en
dc.description.statusPeer-revieweden
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationCarlos-De Clercq, N., Breen, L. and McCarthy, T, (2023) 'Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration', UCC Student Medical Journal, Vol. 3, pp. 14-25. https://doi.org/10.33178/SMJ.2023.1.2en
dc.identifier.doihttps://doi.org/10.33178/SMJ.2023.1.2
dc.identifier.endpage25
dc.identifier.issn2737-7237
dc.identifier.journalabbrevUCC SMJ
dc.identifier.journaltitleUCC Student Medical Journalen
dc.identifier.startpage14
dc.identifier.urihttps://hdl.handle.net/10468/16559
dc.identifier.urihttps://journals.ucc.ie/index.php/smj/article/view/4319
dc.language.isoenen
dc.publisherUCC Medical Research and Technology Societyen
dc.relation.urihttps://journals.ucc.ie/index.php/smj/article/view/4319/6485
dc.rights© 2024, the Author(s). This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.en
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0
dc.sourceBatch uploaden
dc.subjectPeripheral nerve injuryen
dc.subjectNerve regenerationen
dc.subjectRegeneration associated genesen
dc.subjectGAP-43en
dc.titleDesign and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regenerationen
dc.typePeer-reviewed Articleen
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