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Mouse whole embryo culture and the analysis of neural tube defects

Culshaw, Lucy; (2019) Mouse whole embryo culture and the analysis of neural tube defects. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Whole embryo culture enables direct observation and manipulation of organogenesis stage embryos, that would otherwise be relatively inaccessible within the maternal uterus. Rat serum is the primary medium for mouse embryo culture and can sustain growth and development comparable to that in utero. To enhance the “replacement, reduction and refinement” (3Rs) of animals in research, culture experiments were performed to determine whether serum-free medium can substitute for whole rat serum, or whether rat serum can be diluted, and yet still maintain high quality development in vitro. Of two serum-free media tested, neither could sustain development comparable to that achieved in 100% rat serum. However, dilution of rat serum 1:1 with a defined medium supported growth and development with no significant differences from 100% rat serum. Hence, rat usage can be reduced by culture in medium containing diluted serum. Neural tube defects (NTDs) are severe birth defects of the brain or spinal cord. NTDs occur in mice lacking ASPP2, a p53 agonist and tumour suppressor. Embryos with a deletion of exon 3 in the ASPP2 gene, Trp53bp2, were found to have a progressive NTD phenotype that worsened with gestational age. The Trp53bp2Δ3/Δ3 embryonic neural tube phenotype involves ventral neuroepithelial overgrowth, ectopic lumen formation and re-opening of the neural tube. Cellular analysis revealed disruption in Trp53bp2Δ3/Δ3 embryos with and without a macroscopic phenotype. These histological abnormalities included disrupted proliferation, disorganised differentiation and a reduced basal to apical migration of cells prior to mitosis. Using the open-yolk sac embryo culture method, a hypothesis based on over-proliferation in Trp53bp2Δ3/Δ3 embryos was tested using the chemical inhibitor DAPT. It is proposed that deletion of ASPP2 results in an apico-basal polarity defect which increases in severity through development and results in rupture of the neural tube at variable locations along the body axis.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Mouse whole embryo culture and the analysis of neural tube defects
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10068473
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