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Title: Gene targeting studies at the mouse prion protein locus
Author: Moore, Richard C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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The prion protein (PrPc) is a normal host-encoded glycoprotein which accumulates as a disease specific protease-resistant isoform (PrPsc) in the brains of infected hosts. In a number of species PrP polymorphisms and germline mutations are associated with the modulation of disease phenotype and the occurrence of familial prion disease. To investigate the biological consequences of manipulation of the prion protein in mice a flexible two step double replacement gene targeting strategy was developed. This method can be used to generate a series of mouse lines with alterations to the mouse prion protein gene (Prn-p). To facilitate gene targeting studies a restriction map of the 129/Ola Prn-p locus was constructed and a series of overlapping genomic clones were retrieved from a λ DASH II bacteriophage 129/Ola library. The double replacement strategy was used to generate PrP deficient mice and mice with subtle alterations to PrP codons 108 and 189. Murine PrP 108F/V_189L/T dimorphisms give rise to 2 distinct PrP allotypes, PrP-A and PrP-B and these are postulated to be responsible for the control of incubation time following challenge with a wide range of prion inocula. To test this proposal the endogenous 129/Ola PrP-A allotype [108L_189T] was converted by gene targeting to encode the PrP-B allotype [108F_189V]. Mice bearing codon 108 and 189 alterations were challenged with mouse adapted BSE isolate 301V. Gene targeting in 129/Ola derived HM-1 ES cells and breeding with 129/Ola mice enabled the investigation of the effect of PrP alterations in the absence of PrP overexpression artefacts or the influence of non-Prn-p genes. The dramatic acceleration of incubation time in mice homozygous for the Prn-pa[108F_189V] gene targeted allele confirmed the major role of codons 108 and 189 in the control of BSE isolate 301V incubation time - and probably other prion isolates. This data provides the strongest evidence yet that the incubation time control, long attributed to the action of different alleles of Sinc (Prn-i), is determined by PrP codon 108L/F and 189T/V dimorphisms.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available