3 replies to this topic

[Andras]

I am experiencing some problems with one of my projects right now. I have a viral protein that I'm trying to have HEK293s expressed. I have an mCherry tagged version in a plasmid, which is transfected into the cells, and there's a G418 resistance gene on this plasmid. (The HEK cells do not have the large T antigen.)
First of all, even though the cells do not have the antigen, 1mg/ml of G418 is not killing all wt -they survive, but their growth is really slow compared to the others'.
My real issue is: even though the cells in general are red under a microscope, when I collect protein, and do a Westerns for the viral protein, the expression levels are varied AND low. It seems like that the cells are losing the protein (or downregulating its expression).
That, of course, means that I can't do any Westerns for my proteins of interest, which we suspect might or might not be affected by the presence of this viral protein... I need to make these guys express this protein, in a relatively stable manner, otherwise the whole project is useless.
How do people go around this issue? I don't want to reinvent the wheel, and I'm running out of time for my PhD... I read a lot, but I would like to ask people as well, because I don't have a great deal of experience with creating and working with stably expressing cell lines. (While we're at it -it was a great surprise to me when I started the program, that eukaryotic cells can express plasmid encoded proteins.)
I thought about cloning the construct into lentiviral vectors, and integrating the protein into the genome, but we had experience with GST tagged versions, and even those guys got downregulated. For two months I've been doing Westerns before I realized that the antibodies work, I just have no protein...
Thank you for your time.

Edited by Andras, 05 August 2011 - 06:52 AM.

[doxorubicin]

It sounds like you could re-transfect and try higher doses of G418 to be sure the cells without "stable" integration are killed.  It is very odd that you see red cells and yet no expression by Western.  Are you sure this is not just the autofluorescence that even untransfected cells exhibit?  If everything you say is true, you should be able to just sort, by FACS, the bright red cells and grow these up.

[Andras]

doxorubicin, on 05 August 2011 - 02:55 PM, said:

It sounds like you could re-transfect and try higher doses of G418 to be sure the cells without "stable" integration are killed.  It is very odd that you see red cells and yet no expression by Western.  Are you sure this is not just the autofluorescence that even untransfected cells exhibit?  If everything you say is true, you should be able to just sort, by FACS, the bright red cells and grow these up.

Thank you for the answer. I did try higher concentrations (1.5-1.75), but the cells hardly grew. I did FACS as well -but the cells sorted this way are not different from the cells grown in G418. (It's not autofluorescence, either -the non-transfected cells do not exhibit it.)
There are relatively few really strong expressing cells, and a bunch of weakly expressing ones -and I think this is my trouble there...

[asli]

Andras, on 05 August 2011 - 06:51 AM, said:

I am experiencing some problems with one of my projects right now. I have a viral protein that I'm trying to have HEK293s expressed. I have an mCherry tagged version in a plasmid, which is transfected into the cells, and there's a G418 resistance gene on this plasmid. (The HEK cells do not have the large T antigen.)
First of all, even though the cells do not have the antigen, 1mg/ml of G418 is not killing all wt -they survive, but their growth is really slow compared to the others'.
My real issue is: even though the cells in general are red under a microscope, when I collect protein, and do a Westerns for the viral protein, the expression levels are varied AND low. It seems like that the cells are losing the protein (or downregulating its expression).
That, of course, means that I can't do any Westerns for my proteins of interest, which we suspect might or might not be affected by the presence of this viral protein... I need to make these guys express this protein, in a relatively stable manner, otherwise the whole project is useless.
How do people go around this issue? I don't want to reinvent the wheel, and I'm running out of time for my PhD... I read a lot, but I would like to ask people as well, because I don't have a great deal of experience with creating and working with stably expressing cell lines. (While we're at it -it was a great surprise to me when I started the program, that eukaryotic cells can express plasmid encoded proteins.)
I thought about cloning the construct into lentiviral vectors, and integrating the protein into the genome, but we had experience with GST tagged versions, and even those guys got downregulated. For two months I've been doing Westerns before I realized that the antibodies work, I just have no protein...
Thank you for your time.

Hello.
I would suggest doing a "G418 kill curve" with your cells to determine the concentration causing the sharp drop in cell number, this can be quantified by MTT or trypan blue dye exclusion test. Your cells might be less sensitive to antibiotic, I mean less than what is reported in the literature.
Coming to stable transfections, this is how we do it with mammalian expression vector pcDNA3.1 :  treat the transfected cells and control wt cells as well with that concentration of G418 on the same day, passage them to larger (100mm) plates and continue treatment until all control cells die. This ensures that all cells that survive have the plasmid integrated into their genome. In this time period, transfected cells also die severely but not comletely. We then reduce the antibiotic conc. to half, to let the transfected cells grow and form colonies. We either select monoclones or trypsinize and collect all cells as a polyclonal population.
I suppose your transfections are transient. Might want to switch to a stable transfection system. İt takes longer to select the transfectants, but after that you can freeze down the stably transfected cells, reducing time required for each transient transfection. Plus, might improve the situation with protein amount variation.

Hope this helps.