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Title: A transgenic approach for investigating the role of WNK4 in pseudohypoaldosteronism type II
Author: Chowdhury, J. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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Pseudohypoaldosteronism type II (PHAII) or Gordon Syndrome, is a rare familial monogenic disorder caused by mutations within the novel serine-threonine kinases WNK1 and WNK4. I describe here the development of a novel tetracycline-inducible transgenic mouse for PHAII in which the transgene carrying cDNA for a known disease causing mutation in WNK4 (Wnk4 Q562E) is regulated by the Tet-On system. The transgene contains a fluorescent marker a transactivator and a tetracycline response element, all in one plasmid, responsible for driving the expression of Wnk4 Q562E cDNA in the presence of doxycycline. I show the steps taken in the creation of a transgenic PHAII animal model which resulted in six animal lines being produced with differing copy numbers of the transgene. The low, the middle and the highest transgene copy number animal lives, were chosen for further characterisation and then bred to homozygosity. All three chosen homozygous Wnk4 Q562E transgenic animal lines overexpressed the mutant, Wnk4 Q562E, mRNA transcript and exhibited the phenotypes of high blood pressure and hyperkalemia when dosed with doxycycline. Reversibility of the model and tight regulation of the transgene in these animal lines was demonstrated when doxycycline was removed, which resulted in a reduction in Wnk4 Q562E mRNA levels and a return to normal blood pressure and normal plasma potassium levels. I also show by Western blotting microdissected segments of the nephron from doxycycline treated mice, that expression of NCC is increased as expected in the DCT. Increased NCC phosphorylation was also observed, suggesting increased transporter are responsible for the observed rise in blood pressure. FISH was used to visualise the presence of the Wnk4 Q562E transgene construct within one of these animal lines; the transgene was located on chromosome 4.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available