COS-7 and PC-3 transfected with a GFP-AR fusion protein - (Dec/21/2006 )
Hello to everybody out there!!!
Recently I tried out a few transfection reagents for transient transfection of COS-7 and PC-3 cell lines. We received high tranfection efficiency with the new FuGeneHD (around 70-80%). The cells have been transfected with a pEGFP-C1 plasmid encoding for a GFP-hAR (androgen receptor) fusion protein under the control of a CMV promotor. Although, I am quite satisfied with the high efficiency I still observed a few problems when analysing the cells under the microscope.
The main problem is that I have some highly gfp-emitting "dots/vesicles" inside the cytoplasm (or perinuclear). Nevertheless, not all of the cells are carrying these "vesicles". Might these "artefacts" be inclusion bodies? Cell-cylce dependent (as just around 30% are showing this picture)? Some sort of autophagy because of the high expression of the protein? I don't think that it might be due to mycoplasma contamination.
As far as I do remember I never obsered this when transfecting with calcium phosphate...but as the efficiency for this method is more than just low for COS-7 and PC-3 I had to find another transfection reagent.
Did anybody observed something similar? I am very grateful for any help or suggestions =)
Thanx a lot in advance!
It may be expression-related, or the distribution of your fusion inside cells, or could be transfection process-related. EGFP does have negative effect on cells. You would see the same pattern that you have observed in some, but not all cells using an EGFP vector without fusion.
yes I do think as well that I would observe the same pattern when transfecting the cells with the empty vector...I just wonder if it is due to fugeneHD or the lipofectamine 2000 itself. Probably I could get rid of these "artifacts" when shortening the incubation time during tranfection.
Is high transfection efficiency essential part of your study? if not, say , you only want to determine the location of these fusion proteins in individual cells, you can always use less DNA and lipid reagent dosages to turn the level of expression down, hence any toxicity associated with this process will be reduced as well.
the high efficiency is not that essential as I will analyse the distribution of the fusion protein with a laser scan cytomer which should allow me to analyze a few tousand cells per slide. to use less dna and reagent is a good idea to start with. thanks a lot.