I performed transient transfection of HeLa cells using Lipofectamine overnight, with a GFP-tagged protein. Seen under a confocal fluorescence microscope, one could clearly see cells with good expression of the protein of interest, considering of course that some cells will also not be transfected at all. My question is, will it be possible for a cell to be transiently transfected but only express the protein marginally (almost to undetectable level, detected via fluorescence microscopy), or if transient transfection usually is all or nothing, i.e. once successfully transfected, cells should express the protein significantly (at least most of the time)?
Thank you in advance for your time.
Hey Rafael,
we recently succeeded in transiently transfecting macrophages (peritoneal macrophages, bone-marrow-dervied macrophages) as well as MEFS with mRNA (GFP as a control, but also other mRNAs) and we observed that it depends on the cell type you are transfecting and the properties of the mRNA. Transfected mRNAs undergoe like endogenous mRNAs normal turnover and the cell will fine-tune the levels of the protein, which the mRNA is encoding and the protein, which already exists in the cell. Of course this should not be the case for proteins that are not naturally expressed in the cell like GFP. But also for GFP we saw that the enconding mRNA is expressed much longer in BMDMs and MEFS than in peritoneal macrophages and with a higher transfection rate.
So in my opinion it is not "all or nothing" but like always in biology it highly depends on your cellular context.
If you want, you can have a look at the little paper, we published about that topic:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA, November 2019, Journal of Visualized Experiments
I hope that helped you a little.
All the best,
Marc
In my experience transiently transfecting GFP-tagged proteins and looking at them under the scope, there is a spectrum. I was transfecting GFP-tagged Akt, which is a regulator of many cellular processes, so the cells that were expressing Akt a lot had major morphological changes whereas the cells expressing Akt a little less looked more normal. I ended up imaging the cells that were in the middle (i.e., not super bright) because I didn't want too much of a departure from the regular physiology of the cells, and I also didn't want to burn my confocal detector 😂
Since it is a protein that you transfected, how much fluorescence you can see under the microscope depends upon the transfection efficiency(i.e based on transfection method, cell type variability in transfection efficiency.) . Measuring fluorescence by FACS is also a good way to detect low signals using microscope
Rishi is correct, there will be a spectrum of non-transfected, low, medium and high generally when you transiently transfect cells. remember you are usually expressing your protein of interest at very high levels now, so which level of transfection you need to study depends on your experimental question. Of course, seeing fluorescence in a cell does not always mean the protein is localizing correctly, sometimes the protein can get stuck in the ER. So in addition to being aware of the level of expression you also need to consider is your protein localizing where it is suppose to (or some other mark of proper expression).
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