Transgenic modelling of a novel inherited prion disease associated with PRNP Y163X mutation
Aim
The aim of this project is to study the unusual properties of a newly discovered PRNP mutation, Y163X, using novel transgenic and knock-in mouse models, to investigate the disease mechanism causing this unique prion disease.
Methods
The unusual prion disease properties of Y163X mutation will be investigated by a two-pronged approach: transgenic mice with over-expression of human PrP and human PrP knock-in mice. The key questions we aim to address in these models are: (1) to what extent is the unique Y163X phenotype due to loss of PrP glycosylation or the loss of GPI-anchor as a result of the truncation; (2) to study the relationship between peripheral pathogenesis and neuroinvasion; (3) to test transmissibility of Y163X patient brain homogenates since initial transmission experiments in transgenic mice expressing wild type human PrP were inconclusive, but there is precedent from our previous work that mice expressing the homologous mutant protein may be required to demonstrate transmissibility of some inherited prion diseases; (4) to investigate if we can prevent prion neuroinvasion with small molecules and monoclonal antibodies.
Rotation project
The Transgenic Research Group specialises in generating transgenic mouse models for prion research, and have published extensively on animal modelling of prion diseases, most notably modelling of the kuru resistance gene published recently in Nature in 2015 (Nature 522: 478-481). The student will benefit from our considerable experience by being introduced to an array of techniques and disciplines ranging from molecular biology to reproductive biology employed in the generation and characterisation of transgenic mouse models. The student will learn how to design and undertake preparation of transgene constructs as well as other molecular biology techniques necessary for successfully generating and characterising transgenic mice. The student will also prepare microinjection grade DNA, and inject it into mouse embryos. During the 3-month rotation period, the student will gain a complete overview of the requisite skills for genetically engineering mice for research.
PhD project
The student will start their 3-year PhD focussing on the Y163X transgenic mice that we have already generated and are over-expressing human PrP Y163X mRNA. The Y163X transgenic mice will be crossed with wild type huPrP-expressing transgenic mice, to study ageing cohorts of heterozygous and homozygous mice for any possible spontaneous disease, as well as challenging them with a panel of human prions. The transgenic mice will be analysed by biochemical and immunohistochemical techniques to investigate how well these animals recapitulate the disease features of affected families.
Y163X knock-in mice will also be generated by knocking-in the human PrP open-reading frame with the Y163X mutation by combining CRISPR-Cas9 with single stranded oligodeoxynucleotides (ssODNs) technology.
The disease being modelled has an unprecedented phenotype and as well as offering the student a rounded experience in transgenic research that would be applicable to any other neurological disease, the characterisation of Y163X models will have fundamental implications for prion disease mechanisms, in respect of the role of the GPI anchor and peripheral tissues.
References
(1) Asante, EA and Collinge, J. Transgenic studies of the influence of the PrP structure on TSE diseases. Advances in Protein Chemistry, Book Series Volume: 57 Pages: 273-311 Published: 2001 (2) Mead S et al, A novel prion disease associated with diarrhoea and autonomic neuropathy. N Engl J Med. 2013 Nov 14; 369(20):1904-14. doi: 10.1056/NEJMoa1214747. (3) Yoshimi et al, ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Comms 7:10431 doi: 10.1038/ncomms10431.