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Title: Developing methods to generate single-sex litters
Author: Douglas, Charlotte Louise
ISNI:       0000 0004 9351 3744
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2020
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Producing single-sex litters can have a positive impact for many applications, including reducing disease-spreading vectors, increasing effective breeding populations for conservation, in agricultural biology, and in research; reducing unnecessary animal culling, in accordance with the Home Office 3Rs. In the laboratory mouse model, Mus musculus, males carry the sex chromosome complement XY. The daughters inherit the X chromosome, and the sons inherit the Y chromosome. In this thesis I exploit the unique inheritance of the X and Y chromosome from the male heterogametic sex to investigate multiple methods of generating all-male or all-female litters. Co-inheritance of two alleles, one from the father and one from the mother, which only upon the presence of both alleles causes embryonic lethality, was called a bi-component system. I first performed a proof-of-principle strategy utilising two pre-existing mouse models, X-Cre and an inducible Rosa26-DTA. Diphtheria toxin A (DTA) expression was only active upon excision of a floxed-STOP cassette by Cre recombinase. Co-inheritance of the XCre allele from the father, and Rosa26-DTA allele from the mother resulted in activation of the DTA and female-specific embryonic non-viability. In an alternative approach, I used CRISPR-Cas9 to generate mutations in a target housekeeping gene by co-inheritance of a Cas9 and sgRNA transgene. I first investigated the mutation efficiency of multiple sgRNAs in vitro, targeting an essential gene Topoisomerase 1. Highly-mutagenic sgRNA constructs were then carried forward to generate sgRNA-expressing mouse lines via recombination-mediated transgenesis. The sgRNA-expressing mice were mated with a pre-existing Rosa26-Cas9 mouse line. I showed that co-inheritance of the Cas9 and sgRNA transgene induced mutations in the target Topoisomerase 1 locus. Co-inheritance of the two transgenes also resulted in embryonic non-viability due to loss-of-function of Topoisomerase 1, whilst inheritance of a single transgene did not result in loss-offunction mutations. Lastly, in order to generate sex-specific mutations by CRISPR-Cas9, I generated sex chromosome-linked Cas9 transgenes. I showed that the X-Cas9 transgene expressed Cas9 in vitro and in vivo, and in the future this line may be a useful tool for generating mutations in a target gene in a sex-specific manner.
Supervisor: Turner, James ; Tucker, Abigail Saffron Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available