I'm hoping that someone might be able to answer a problem I'm having. i'm trying to add a GFP tag to my protein of interest in situ in the genome using TALENS and a GFP/puro donor plasmid.
The problem I'm having is that my puro resistance protein is effectively under the same transcriptional control as my protein of interest as the GFP is replacing the stop codon, and the puro resistance gene is separated from the GFP by a 2A linker. My protein of interest is expressed only in S-phase though. So my question is, will the puro resistance protein be stable enough in the cell to allow for selection of those cells that have the correct integration?
Thanks in advance for any help or advice you might be able to spare!
All the best,
I would think that the stability of the PAC is only half of the equation. The other half would be how active the promoter is during the S-Phase and whether the targeting vector that carries the GFP-2A-Puro also expresses on its own. If the vector is capable of expressing the PAC before recombination at the TALEN's cut site, then you can do a pulse selection of two days to increase the number of stable integrants. I've seen a couple papers that used Puromycin to select for transient resistance (one included below) but they used a higher lever promoter to do this. If your targeting vector does not express on its own, then you'll probably have to find out through trial and error. Worst case scenario, you have to flow-sort for GFP-positive cells.
"Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: A transient gene-integration marker for ES cells"