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Title: Mouse whole embryo culture and the analysis of neural tube defects
Author: Culshaw, Lucy
ISNI:       0000 0004 7660 9189
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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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.
Supervisor: Copp, A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available