Help with transfection of HEK cells with p53 flag - (Mar/04/2008 )
I have a very strange problem. I need to express exogenous p53 in HEK cells. Everything is fine, I'm getting huge fold increased in both protein and mRNA level. The only problem is when I'm trying to split the cells. It happened many many times. When I make lysates or RNA from the plate which was transfected everything looks good, but I wanted to split transfected cells for further drug treatment assays. And here is the problem, after that they are just dying, if I'm not touching them, everyhting is fine, but when trying to split 48h after transfection, the are dying. Is it because of p53 or what? Please help me! if for the final experiment i need at least 20 transfected plates, why I can just make 2 and then split them in ratio 1:10??????????????
Not sure what are you trying to do, just for saving time and $$$? Your cells die because of many reasons. It could be due to toxicity of the transfection procedure, or it could be due to the expressed protein together with cell proliferation. Can't you use transfected cells, why do you split cells after transfection? You lose DNA per cell in a big way for each passage, unless you are using EBV replicon based plasmid.
well i was thinking that after transfection of 1 plate i can split cells into 5 plates and after that start the treatment?
But the problem is that the protein you are overexpressing controls the cell cycle! And since p53 will induce the production of p21, that will stop the cell cycle and eventually induce apoptosis.
p53 is a major trigger for cell cycle arrest and apoptosis. Once the cells become unattached from the plate via trypsin for splitting they may not be able to reattach and hence die. Why not just seed out the plates you want for the final experiment at a lower density and transfect these plates? Granted 20 plates is a lot of reagent and DNA but it may be the best way to get the final desired product. Otherwise, I have done transfections during splitting and had good results. The only other idea I have for you is to create a tetracycline-controlled stable cell line. This will take a bit of work up front but may actually be a huge advantage in the end. Invitrogen sells HEK293 cells in their Flp-In Trex system. This would allow you to make a line very quickly. Otherwise, check the literature to see if anyone has published a cell line that you could use. I know Agarwal et al. (Cancer Research, 2007; 67:(1)) has published a TR9-7ER cell line that is tet-off for p53 expression but I'm not sure if this line would suffice for your needs.