TROPLIST: Brightest Fluorescent Proteins

[Mustafa Khokha]

So I wanted to post a follow-up on some questions regarding brightest  
fluorescent proteins (FP's) in Xenopus
that I received on and off line.  Just to recap - I injected various  
fluorescent protein RNAs into 1 cell of a 2 cell
embryo and then waited about 24 then 48 hours and scored them for  
brightness using a fluorescent stereoscope
and my eyeball as brightness indicator.  The filter sets are for GFP  
and rhodamine.

For the GFP filter,
YFP appeared brightest.  Next was GFP which was a close second.  Both  
of these were brighter than
Venus.  And all of these were much brighter than mCitrine.

For the Rhod filter
tdTomato was the brightest clearly.  Then mCherry and RFP were  
similar and bright.  They were all
much brighter than mStrawberry.  mOrange was weak.

A number of questions surfaced:

1.  all of the FP's were cloned into either CS2 or CS108 and  
therefore transcribed with Sp6 using the Ambion
mMessage machine kit - with cap, etc.  There were no b-globin UTRs or  
other UTR's other than the multiple
cloning sites for CS2 or CS108.  I linearized all the constructs 3'  
to the SV40 Poly A signal so that should also be the same
when making RNA.  YFP, GFP, Venus, RFP were in CS2, the others were  
all in CS108.
Has anyone noticed any difference between CS2 or CS105,7,8 with  
regard to protein product?

2.  I did all the cloning for mCitrine, tdTomato, mCherry,  
mStrawberry, and mOrange (all of which are from Roger Tsien's lab)
which is basically just the coding region cloned into CS108.  I  
sequenced GFP, RFP, and YFP.  GFP was the eGFP variant.
YFP was standard YFP (venus is a YFP variant and supposed to be  
brighter). Of note, the RFP is a membrane tagged version.
None of the others are tagged in any way.

If anyone has any further suggestions, I'd love to hear them.  I  
think it is important to resolve this issue.  I'll probably remake  
the RNAs
and try injecting them all again to confirm the result.  If anyone  
has any other suggestions or results that are similar/differnt that  
would be helpful.

Mustafa