I added a NLS (nuclear localization sequence) to my clone of interest and expressed it in my cells. However, the protein when expressed in the nucleus of the transfected cells is causing the cells to die in large numbers. I am not sure whether this is due to the NLS (I have previously created a stable line of the protein without the NLS and there was no problem) or the protein upon entering the nucleus is causing apoptosis?
In either case, how can I now generate a stable line for the NLS-expressing plasmid?
Hi,
You need to use an inducible system.
For example a Tet-on system.
In presence of tetracycline (or doxocyclin), your plasmid will express your construct. Without doxo, there will be no expression theorycally. In reality, there is a weak leak of basal expression.
It's been a while I haven't used such system so you'll need to check about uptades and system improvements.
Alexis
The apoptosis may result from ectopic overexpression of your target gene controlled by a strong promotor of your expression vector (so-called your construct). You can try knocking NLS-coding sequence into the place just after the start codon of your interested gene to make it expressed as normal but just in nucleus by EET technology and observe whether the cells die in large number or not. Good luck!
Hi Rituparna. One of the best inducible systems I have used is generated by fusion of a portion of the murine Oestrogen receptor to the protein of interest (attached to 3' end of gene).
Addition of Tamoxifen activates the protein (it binds the ER section and removes the ER's steric hindrance). If you can get a hold of this construct (I don't have it I'm afraid)
you could construct a NLS-protein-ER fusion which should work.
Sorry I can't be more help,
Gary
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line