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Type: Thesis
Title: Mouse genome modification and investigation of episodic disease
Author: Robertson, Louise Jane
Issue Date: 2018
School/Discipline: School of Biological Sciences
Abstract: Mouse models are essential tools for biomedical research, allowing researchers to investigate gene function and model human diseases. The mouse genome can be manipulated to ablate (“knockout”) gene function, create targeted point mutations or insert transgenes or small epitope tags. Previous gene targeting methods for creating these modifications were slow and inefficient. The arrival of CRISPR/Cas9 genome editing technology has revolutionised the production of genetically modified mice as it is easy to use, efficient and cost-effective. The research comprised in this PhD thesis explores the use of genetically modified mice to better understand human diseases, as well as developing new technologies for mouse genome editing. The first manuscript describes a phenotypic investigation of the transgenic Prrt2 knockout (Prrt2 KO) mouse. In humans, mutations in PRRT2 cause an infantile epilepsy syndrome (benign familial infantile epilepsy; BFIE) and a movement disorder in adolescence (paroxysmal kinesigenic dyskinesia; PKD). We identifed a spontaneous paroxysmal phenotype in Prrt2 KO animals, as well as premature death in HET and KO mice. Behavioural tests also revealed learning deficits and gait abnormalities in KO mice that may reflect phenotypes in homozygous patients, confirming the utility of this model for investigating PRRT2-related disorders. The second manuscript examines variants of the Streptococcus pyogenes Cas9 endonuclease (WT SpCas9) commonly used for CRISPR genome editing. Numerous Cas9 variants have recently been characterised, each recognising different PAM sequences. Most of these variants remain untested for genome editing in mice. In this study, we tested a selection of endonuclease variants (SpCas9 VQR, SpCas9 VRER, SaCas9 KKH and AsCpf1) for their ability to edit the mouse genome via mouse zygote injection, with the aim of expanding PAM targeting options. We showed that all variants are able to mutate the mouse genome, albeit with different efficiencies. We also highlighted the propensity of SaCas9 KKH to generate heterozygotes or mosaic offspring in which at least one allele remains unmodified. When editing using a ssDNA oligonucleotide repair template, SaCas9 KKH consistently left a wild type allele in correctly targeted offspring, whilst WT SpCas9 frequently mutated the other allele. This characteristic could be beneficial when targeting genes in which nullizygous mutations cause embryonic lethality, as the high efficiency of WT SpCas9 commonly prevents the production of viable offspring. With CRISPR/Cas9 technology, it has become quick and efficient to tag endogenous proteins with small epitope tags. The third manuscript in this thesis compares a series of commercially available antibodies for their efficacy to detect epitope tags on Pcdh19 HA-FLAG expression in mouse brain tissue. This model was generated within the laboratory to allow specific staining of PCDH19 for investigating protocadherin 19 girls clustering epilepsy. Of the 8 antibodies tested, only two (one HA and one FLAG) were specific for PCDH19. This data will provide guidance for researchers designing similar studies, preventing extensive optimisation of immunofluorescent staining. Together, the data presented in this thesis demonstrate the versatility and importance of mouse models for the study of gene function and neurological disease. This research provides more options for producing genetically modified mice and streamlines the downstream applications of endogenous epitope tagged genes.
Advisor: Thomas, Paul
Hughes, James
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018
Keywords: Epilepsy
movement disorder
mouse model
epitope tag
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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