Forcing a protein to express in the nucleus.
Is adding the SV40 NLS signal to the N-terminus usually enough?
Excluding a protein from the nucleus.
I’m less sure about how to do this. The protein of interest has a predominantly nuclear localization, but does not have an easily defined NLS. Should adding a fluorescent reporter to the N-terminus eliminate any putative NLS? Has anyone tried adding nuclear export signals to a transgenic protein?
I need to rescue the same 571aa protein in and outside the nucleus with a dsRed tag, and compare phenotypes.
Any thoughts would be greatly appreciated.
The SV40NLS-GFP-lacZ fusion designed in the Fire lab gives a very strong nuclear accumulation - in our hands without any signal left in the cytoplasm. Detailed description of the construct (e.g. pPD96.02) can be found at http://www.addgene.org/labs/Fire/Andrew/Vec95.pdf. It is stated that SV40 NLS-GFP without the lacZ localises to cytoplasm and nucleus, presumably because of rapid diffusion. In your case, SV40 NLS-571aa-dsRed should be sufficently big to stay in the nucleus once imported.
Keeping it in the cytoplasm:
Simply adding a FP to the N-terminus will most likely NOT prevent nuclear import. I’m not aware of detailed analysis of nuclear export signals in C. elegans proteins, but have a look for instance at the following papers:
For strong nuclear localization, we and others have had good results with a histone H2B coding region fused to GFP or mCherry. Another choice might be the putative NLS from EGL-13, - see recent paper by Lyssenko et al. in November 2007 issue of BioTechniques. We haven’t tried it but it looks promising.