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Ethics, R&DGenetic testing
Testing function of genetic variant in Human cells
Functional testing in Ion Channel Arrhythmias
Fluorescent antibodies
Long QTdelayed repolarization of the heart and increases the risk of episodes of torsades de pointes
LQT-
Brugada syndrome-
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
KCNQ1 (676aa, 74.7kDa)
KCNE1 (129aa 14.7kDa)
SCN5A (2016 aa, 227kDa)
KCNH2 (1159aa, 126.7kDa)
Construct wildtype
Action potential
SCN5A + Nav1.5
KCNH2 - Kv11.1 (I kr)
KCNQ1 - Kv7.1 (Iks)
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
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).
Site specific mutagenesis
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
Confocal MicroscopyKCNQ1 RedV5 Green Nuclear Blue
Potassium fluxOR assay
Acknowledgments
Julian SampsonDhavendra Kumar
Chris GeorgeAlan WilliamsSam Mason
Peter O’CallaghanCath Owen
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