In a mixed culture of immortalized MEFs (Large Tag), I need a way to kill a *specific* subset of the cells fast (hopefully within 1-2hr). So, I am looking for a drug or treatment that can kill MEFs fast but also to transfect MEFs with some kind of resistance gene so I would get a selective killing of only the untransfected cells in the mixture (or vice versa- transfect the cells I want to kill with a pre-conditioning gene that would make them highly sensitive to the treatment). I tried G418 but after 3 hrs, there is little death even at a high concentration. That is too slow for me.
Any ideas?
The choice of treatment will depend on the mechanism by which it acts on the cell, which in turn is linked to the 'resistance' gene you transfect in. G418 blocks protein synthesis and it takes time for protein turnover, which is why it takes so long for proteins critical for cell survival to be depleted. The second point of consideration to your question is that you want cell death within 1-2h, which would often be necrosis instead of apoptosis, and necrosis is hard to block.
I'd recommend 0.1-1mM H2O2, though I'm not confident that any overexpression of catalase is enough to counter necrosis-inducing dose of H2O2. Instead, you might want to look into ion channel literature for compounds which induce cell death through unregulated ion movement; it should be easier then to block that ion channel's function via transfection
Interesting problem. It's going to be hard to get the expression of your resistance gene up-enough in 1-2h. Let alone complete a whole death / resistance process. Frankly I'd look into sorting rather than killing. For example, with a lightning-fast promoter ("immediate-early" is the word..) you might just get enough GFP up to do a FACS to separate transfected from non-transfected cells. Not sure if this can really work in 2 hours. It's the quickest thing I can think of anyway.
Whatever you do, you're going to be limited by how quickly your transgene expression can kick in at all, and 2 hours in a mammalian system is pushing it.
Hi John,
I tried FACS. The sorting is not the problem. Just detaching the cells from the plate (e.g. trypsin) causes the cells to loose the molecules I want to follow.
If you are looking for cell death within 1-2 hours of a specific point (not 1-2 hours post transfection) you could transfect the cells with a photosensitizer (e.g., KillerRed from Evrogen) that has a NLS. Light exposure (green-yellow wavelengths) can then be used to cause cell death.
Timothy - this is an EXCELLENT idea. I had no idea such proteins existed. Thank you very much.
Hi Gal,
although I can not help you with an answer to your question this is really an interessting problem. I wish you all the best and would ask you to led me know if the transfection with photosensitizer had helped to solve the problem.
If anyone still follows...
I tried KillerRed. In my hands, my cells (immortalized MEFs) two types of promoters (CMV or Ubc) and several microscopes for variable exposure times (2->20 minutes) - didn't work (cells just didn't die).
I am now trying diphtheria toxin to selectivly kiil human cells in a mouse-human co-culture. so far, some cell lines are resistant (U2OS) and the others just die too slowly for my purposes (H1299 ~50% after 30hours, HEK293, HEK293T ~10-20% still alive after 30hour).. But, am still trying..
If anyone is interested, I found a way. Its called Raptinal - a new drug that induces apoptosis and kills >99% of the cells within 1hr!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683402/
APAF-1 knockout cells are somewhat resistant (i.e. they survive for at least 2hr, then die, probably necrosis through another pathway). So I can do a co-culture of APAF-1 k/o + WT cells and kill the WT cells before the APAF-1 cells die. Hopefully, I'll get good results for what I'm looking for (doing the experiments now).
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