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Title: Targeted mutagenesis of murine Scn5a : generation and characterisation of a mouse carrying the δKPQ mutation
Author: Head, Catherine Elizabeth G.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Mutations in SCN5A, the gene encoding the α (pore-forming) subunit of the voltage-gated cardiac sodium channel, have been implicated in three important arrhythmia syndromes: long QT Syndrome (LQT3), Brugada Syndrome and progressive cardiac conduction defect. In Brugada Syndrome the mutations are loss of function, whereas in LQT3 they are gain of function, resulting in a late sodium current and hence prolongation of the action potential. The aims of this project were to generate and characterise gene-targeted mouse models of the Brugada Syndrome (null mutation) and LQT3 (ΔKPQ mutation). The N5 mouse line was generated as a potential Scn5a null using embryonic stem (ES) cell clones in which the recombination event appeared to be an insertion of the construct, rather than replacement. Analysis of the offspring of subsequent heterozygote intercrosses suggested that the integration was not a targeted insertion but a so-called “type III” recombination event, in which the targeting construct had integrated near the centromere of a non-homologous chromosome. This was confirmed by fluorescence in situ hybridisation (FISH). To generate the Scn5atm2Cam mouse line the ΔKPQ mutation was introduced into the genome by gene-targeting with positive-negative selection n ES cells. Male chimaeras transmitted the ΔKPQ mutation to 50% of their offspring. No homozygote offspring were born from heterozygote matings: embryonic lethality occurred around 10.5 days. Heterozygote frequency was less than Mendelian at six weeks but no later excess mortality was observed. Delayed repolarisation was shown by an increase in ventricular effective refractory period and APD90, but there was no significant change in the surface ECG. The expression profile of Scn5a was found to be more extensive than previously thought. Other voltage gated sodium channel α-subunit isoforms were also shown to be expressed in the heart.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available