ethics, r&d genetic testing testing function of genetic variant in human cells functional...
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Ethics, R&DGenetic testing
Testing function of genetic variant in Human cells
Functional testing in Ion Channel Arrhythmias
Fluorescent antibodies
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Long QTdelayed repolarization of the heart and increases the risk of episodes of torsades de pointes
LQT-
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Brugada syndrome-
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The Ion channel genes that can cause LQT or Brugada Syndrome:
KCNQ1KCNH2SCN5AKCNE1KCNE2
70-80% of LQT mutations are in these genes &20% of Brugada mutations are in SCN5A
The flux of ions across membranes is responsible for the generation of the action potential
}} Β-subunits
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KCNQ1 (676aa, 74.7kDa)
KCNE1 (129aa 14.7kDa)
SCN5A (2016 aa, 227kDa)
KCNH2 (1159aa, 126.7kDa)
Construct wildtype
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Action potential
SCN5A + Nav1.5
KCNH2 - Kv11.1 (I kr)
KCNQ1 - Kv7.1 (Iks)
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The model
To express channels of interest in human cells Flp-In 293using vector pcDNA5/FRT/V5-His TOPO
Except KCNE1 in mammalian blasticidin vector
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Transfection into Flp-In cell system1. Allows integration and expression of your gene of
interest in mammalian cells at a specific genomic location.
2. Involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice (Flp-In 293).
3. The gene of interest is then integrated into the genome via Flp recombinase mediated DNA recombination at the FRT site (O'Gorman et al., 1991).
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Site specific mutagenesis
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MutagenesisKnown gene variants identified can be introduced and compared to wildtype for RISK STRATIFICATION
KCNQ1, - 11 variants constructed and 6 transfected with beta-subunit*
KCNH2- only wildtype.
SCN5A – 2 variants constructed and transfected. 1. R1623Q results in a gain of function
related to LQT3 2. S910L results in a loss of function
related to Brugada syndrome
*KCNE1-wild type transfected as beta-subunit for KCNQ1
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Confocal MicroscopyKCNQ1 RedV5 Green Nuclear Blue
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Potassium fluxOR assay
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Acknowledgments
Julian SampsonDhavendra Kumar
Chris GeorgeAlan WilliamsSam Mason
Peter O’CallaghanCath Owen