What transfection method are you using? Stable/transient? What kind of chemicals do you use?

Stable transfection with calcium-phosphate as method. In general transfection method is not problem in the lab since other constructs are expressed nicely.

In calcium phosphate method it sometimes helps to try different time intervals from mixing DNA and the reagents to adding the mix into culture medium and also pH of the buffer plays a role, as reviewed by Thomas & Smart (2005; doi: 10.1016/j.vascn.2004.08.014 ). Also I found that commercial transfection reagents can provide higher expression, however they can also affect the membranes so the patches are less stable. In our lab FuGene HD worked pretty well giving decent expression and not affecting the condition of the cells too much.

Ok, thanks for directions. However, since my receptor activation already leads to membrane blebbing, membrane stability and cell condition are very important, since I need cell to be stable for relatively long time.

What promoter is your construct using. The transfection efficiency (and modifying your transfection methods) will affect the number of cells that are transfected, but won't have as much (if any) effect on expression per cell. The promoter you use will affect expression per cell much more strongly.

The construct is with CMV Promoter, and for expression induction I use Tetracyclin ( it is vector pcDNA 5/FR/TO).

With wild type channel there is no problems at all, nicely expressed, but concatamer is relatively big and rigid and I suppose that is the problem. It is expected, of course, that this appears as difference among wild type receptor and concatmer, but I would just like to increase average expression of concatamer a bit.

I was thinking about temperature decrease at 33 during part of incubationa I read in publication or increasing Tetracyclin concentration (actually tried but result was not something thrilling) and to check did anybody had positive experience at least with temperature: not to sound that I do not believe to publication, it is Plos One 2015, Lin and HuangArticle Enhancing Protein Expression in HEK-293 Cells by Lowering Cu...

The solution of letting cells for more days incubation is not quite the option since after 5th day it is almost impossible to find single nice quality cell for measurement. Or maybe that I resuspend them some 2nd, 3rd day as an option.

And the problem is definitely not promoter silencing and passage number, I measured very freshed and compared to wild-type of similair status. Guess something is going on on the path of concatamer to cell membrane.

Dear Milica,

In our lab it is established practice to follow a quite old indication from this publication: Cooper, Sandra T., et al. "Up-regulation of cell-surface α4β2

neuronal nicotinic receptors by lower temperature and

expression of chimeric subunits." Journal of Biological

Chemistry 274.38 (1999): 27145-27152.

That is to preincubate transiently transfected HEK cells (in our case, with Lipofectamine 2000) at 30°C for at least 12 hours before recording. There's no problem at all with survival or membrane integrity if recordings are made on the third and fourth day after transfection (i.e. transfection on mother plate, dilution of transfected cells and seeding, growth, preincubation at lower temperature and recording). We work with nAChRs, I don't know if in your case the same can be applied, but I've heard rumors that this kind of mechanism may be applied to most heterologously expressed ion channels.

Best,

Thanks alot, Giulia! So I hear some positive experience with this incubation temperature game :)

You're welcome :) yes, for my experimental series it worked very well, hope will do the same for you!