Test of the transient transfection of hERG in HEK293: when to begin

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zhangxu04's picture
Test of the transient transfection of hERG in HEK293: when to begin

I study the hERG K+ channel in transiently  transfectied  HEK293 cells.
I transfect cells at 50-60% confluence on one day after plating. Then I test the currents using  whole cell patch clamp electrophysiology . 
It is suggested to perform the patch clamp experiment on the day after transfection without passaging. But other methods suggest the transfected HEK293 cells should be pasaged on time and then to make the patch clamp (whole cell mode).
I hope someone could give me a suggestion as to when to begin the test and whether it is necessary to passage the transiently transfected 293 cell and then to begin the patch clamp experiment.
thank you very much

Fraser Moss
Fraser Moss's picture
You could do it either way,

You could do it either way, it just depends on what expression levels you need to perform your experiments. I usually wait 48h after transient transfections without passaging to record.
I expect the method that requires passaging is supposed to allow the cells to recover from the transfection and limit any artifact induced from cells still recovering from transfection.
Furthermore, if the cells you are using are actually HEK 293T cells (not just normal 293s) they will be able to replicate the expression plasmid if it has an SV40 origin or replication, increasing the number of cells that are positively transfected with the hERG construct after 1 passage.  It is believed in some circles that letting the cells divide one passage allows the expression levels to normalize across the dish, which is desirable for a consistent data set.
However, do not use trypsin to passage the transfected cells as this will also cleave the hERG at the membrane.  Try a non-enzymatic cell dissociation solution as sold by Sigma or "Cellstripper" as sold by Mediatech. Or just mechanically dissociate the cells and replate them (i.e. bash the flask/scrape the dish).
Lastly there are also protocols out there that suggest placing the cells at 30 degrees for 16-24h before recording as this slows cell division but increases proetin expression and the efficiancy with which the hERG subunits fold, assemble and traffic.  See
Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature.
Chen MX, Sandow SL, Doceul V, Chen YH, Harper H, Hamilton B, Meadows HJ, Trezise DJ, Clare JJ.
BMC Biotechnol. 2007 Dec 20;7:93.
PMID: 18096051

zhangxu04's picture
to frasermoss:

to frasermoss:
 thank you very much. you give me so much valuable suggestion. I appreciate it very much.