I am searching for a plasmid expressing RFP in human cell lines. The RFP should work as a transfection control in flow cytometry experiments. Since I prefer some low-cost solution, I found pcDNA3-mRFP plasmid at Addgene (http://www.addgene.org/13032/). I guess this plasmid is intended for preparation of RFP-fusion preoteins, but is it going to express RFP even without any cloning? A prediction tool showed me that a Kozak consensus sequence is present, but the predictions were not very strong. Please, could you help me?
mRFP is a bit outmoded since other red fluorophores (mCherry, mKate, DsRed) perform much better. In addition, pCDNA3 has been replaced by newer series of vectors (pCDNA3.1, pCDNA3.3) that have improved transient expression.
If you already are committed to mRFP due to other fusion proteins you are using, then it should be OK. However, if you simply want to label your cells with red fluorescence, you might look for a different plasmid.
pcDNA3 is a mammalian expression vector with CMV promoter upstream of MCS, so no need for any cloning. Just transfect your cells and check for efficiency. Select them on G418 if you need stable RFP-expression.
Good luck!
Hi Lucie,
pcDNA3-mRFP will work just fine for what you plan to do, just go ahead. There are many better fluorophores of course but no need to worry about the performance of mRFP as it will do just fine in the context of your experiments. The Kozac sequence Abgene chose is indeed not the best but it will work just fine, so again don't worry about it. In case you really need a much better vector+fluorophore I'm happy to send you a blot with ptagRFP-N vector from Evrogen.
Good luck!
Thank you all for your kind answers.
In fact, I was looking for some more recent fluorophore like mCherry or DsRed (to ensure sufficient brightness even of lower transfection efficiency occurs), but did not find any vectors suitable for us (at least at Addegene's).
@ Lucianno: I would highly appreciate your help if you were so kind and sent me the ptagRFP-N plasmid. Please, could we discuss the details by email? The mine is: [email protected]
Thank you!
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