Hello all,
Lately I have been trying to transfect H1299 Tet-off system or HEK-293 Tet-On system with my proteins of interest which are 2 plasma membrane proteins fused with either GFP (the wild type protein) or mCherry (the mutant type of the aforementioned protein).
I transfect cells (when being 70-80% confluent) with Lipofectamine 2000 for 4-6 hours (no serum or antibiotics in OptiMem medium).
The expected result of these transfections should be green and red fluorescent signal (by using different channels at the fluorescent microscope) on the membrane of my cells (when removing/adding tetracycline in media).
The problem that I face is that have mis-location of my expressed fluorescent proteins. They are expressed on the plasma membrane only at 10-20% of the cells while I mainly see signal inside the nuclei (colored dots) or even on the nuclear membrane.
I do not understand why, even if the transfection takes place, my proteins of interest are expressed on the wrong organelle...
PS. The initial DNA plasmids have been checked for possible errors in the nucleotide sequence.
Any thoughts would be helpful for me.
Thank you,
Ageliki
There could be a variety of delivery mechanisms affecting your cargo as it enters the cell. HEK293 is easy to transfect (scroll down here to see some publications https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) but that does not mean that your complexes will arrive at the desired organelle. It is possible that due to intracellular packaging and regulation (especially of plasmids after transcription) you will get the strange distribution of protein. I would look at previous studies with HEK293 (of which there are a lot) and see if you can find any where they also have images of cells with transfected mCherry/GFP plasmids. That should help you understand the biodistribution of your plasmids and what mechanisms are involved therein.
one possibility is that the fluorescent protein fusion is interfering with protein folding or localization. Have you or others shown that this fusion protein is correctly localized in other contexts?
There could be a variety of delivery mechanisms affecting your cargo as it enters the cell. HEK293 is easy to transfect (scroll down here to see some publications https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) but that does not mean that your complexes will arrive at the desired organelle. It is possible that due to intracellular packaging and regulation (especially of plasmids after transcription) you will get the strange distribution of protein. I would look at previous studies with HEK293 (of which there are a lot) and see if you can find any where they also have images of cells with transfected mCherry/GFP plasmids. That should help you understand the biodistribution of your plasmids and what mechanisms are involved therein.
I am not sure about this idea of “intracellular packaging and regulation...of plasmids”. We express plenty of GFP-tagged proteins in 293T cells without issues. While it’s conceivable that this problem reflects a cell line-specific problem, in my experience it is more common that the fusion is causing the problem.
- Badges
- Science method