I was contacted by a user yesterday looking for a destabilized GFP reporter to monitor promoter repression in addition to promoter activation. This is something I haven’t really looked into much, especially not for use in mammalian cells, so I decided to do some digging. Here’s what I found.
For a while, d2EGFP was the state of the art, presumably because it was sold by Clontech. It was generated by fusing the degredation region of mouse ornithine decarboxylase to GFP and it has a half life of about two hours . A large number of d2EGFP plasmids are available from Addgene. More recent work, combining a related destabilized GFP with a destabilized mRNA, has produced an even shorter half life . A recent paper has produced a GFP with a half life of about seven minutes in S. cerevisiae using an N-degron . Similar work has been done in mammalian cells, but the proteins were only assessed for their utility as proteasome activity reporters, not as transcriptional reporters .
A destabilized red fluorescent protein has been reported, but it is based on an obsolete RFP (DsRed-Express) and has a relatively long half life of about 12 hours .
Now, of course, an interesting alternative to destabilizing the protein itself is using a photoswitchable protein. If you use a protein such as mEos2 that can be irreversibly converted from the green to the red set on exposure to 405 nm light, you can ‘destroy’ all the green protein at any time you choose just by photoconverting it to the red species. Any green protein I see after that must be newly synthesized. I haven’t seen anyone who’s actually done this, however.
- X. Li, X. Zhao, Y. Fang, X. Jiang, T. Duong, C. Fan, C.C. Huang, and S.R. Kain, "Generation of destabilized green fluorescent protein as a transcription reporter.", The Journal of biological chemistry, 1998. http://www.ncbi.nlm.nih.gov/pubmed/9857028
- N. Kitsera, A. Khobta, and B. Epe, "Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure.", BioTechniques, 2007. http://www.ncbi.nlm.nih.gov/pubmed/17824390
- J.R. Houser, E. Ford, S.M. Chatterjea, S. Maleri, T.C. Elston, and B. Errede, "An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae.", Yeast (Chichester, England), 2012. http://www.ncbi.nlm.nih.gov/pubmed/23172645
- N.P. Dantuma, K. Lindsten, R. Glas, M. Jellne, and M.G. Masucci, "Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.", Nature biotechnology, 2000. http://www.ncbi.nlm.nih.gov/pubmed/10802622
- C.W. Yung, T.A. Barbari, and W.E. Bentley, "Integrated non-invasive system for quantifying secreted human therapeutic hIL2.", Biotechnology and bioengineering, 2006. http://www.ncbi.nlm.nih.gov/pubmed/16933326