GCaMP6 now available
Posted in Tips
If you’ve been waiting to jump on the GECI bandwagon, you just ran out of excuses.
GCaMP6, a legitimate breakthrough, is now available at Addgene.
Hat tip to ybot.
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What is the difference between CGaMP6f, CGaMP6m, and CGaMP6s? A quick google turned up nothing, and the SfN abstract doesn’t say.
Contact Janelia for the details.
Short version: S = slow time course, but high affinity; F is faster and lower affinity; and M is intermediate.
The big breakthrough here is that this is ~2X as good SNR as OGB-I. So we can now say that GECIs are BETTER than synthetic indicators. This is for the 6S variant. The 6F and 6M variants are not as good (6M is basically par with OGB-I).
The thing OGB-I still wins in spades is onset time constant. GCaMP6 wins in offset time constant, even with 6S.
I would say unless you have a really specific use justifying 6F, go with GCaMP 6S. It is awesome. (6M really is ultra-niche).
One other thing — the way these improvements were obtained was largely by lowering baseline fluorescence while maintaining the same fluorescence when calcium binds. So beware — if you’re counting on basal fluorescence to give you structural information, this will work MOST of the time, but not nearly as well as previous indicators. It may be wise to, e.g., have a red nuclear marker so you know where cells are (!)
GCaMP6 User: Interesting. I know that this is the case with GCaMP5G. However, are you sure that’s also true for GC6? At least in vitro 6s appears surprisingly bright in my hands. Could you elaborate a little?
Cheers,T
Do you have any suggestion for a red nuclear marker construct (or virus) for cortical neurons.
Cheers
F
I suspect GCaMP6 User meant that some sort of cytosolic marker could be useful, so that the entire morphology is labeled, not just the nucleus, but I don’t want to be presumptuous. For a cytosolic marker, there are several RFPs that are popular, e.g., mCherry and tdTomato.
Hi!
I just made our bicistronic dual color GCaMP6 AAV constructs (mRuby2-P2A-GCaMP6s,f,m) available on addgene.
http://www.addgene.org/browse/article/7731/
(6m should be online soon).
We also plan to have them ready to order from the PennU AAV catalog. THis may take a little longer, but they are on their way.
These tools have been the standard for chronic 2p imaging in our lab for over one year now.
Overall, GC6 levels are lower than with the the monocistronic AAVs. However, I consider this to be a good thing, since we rarely reach the expression level that leads to nuclear GC filling – even over many months. Since the mRuby signal is exceptionally bright and expression is pretty stable after 2-3 weeks we can easily image and refind our cells over many imaging sessions with very stable tuning properties (in VC).
As a bonus: mRuby2 is in the nucleus whereas GC6 is not. This makes structural ROIing of all cells (active or not) much easier, because the G/R ratio provides a pure cellular signal devoid of any other structures (bloodvessels, image intensity gradients etc.). After simple thresholding & watershedding these blobs can then be taken as a seedpoint for further processing like adaptive contour growing to the outer rims of the red cellular signal.
For as long as we don’t have our story published, I simply link to these old images: http://goo.gl/mUv7Uy
The design of the 2A construct follows Szymczak-Workman et al. (2012). We use the P2A serquence with GSG linker since this has been shown to yield essentially complete ‘cleavage’ (Sharma et al 2012). We placed GCamP6s in the back since then only one Amino Acid ( is fused to the N-terminus – mRuby2 therefore gets the longer ‘2A-tag’ (see Szymczak-Workman et al.).
Best,
ybot
—
Sharma, P., Yan, F., Doronina, V. a, Escuin-Ordinas, H., Ryan, M. D., & Brown, J. D. (2012). 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucleic acids research, 40(7), 3143–51. doi:10.1093/nar/gkr1176
Szymczak-Workman, A. L., Vignali, K. M., & Vignali, D. a a. (2012). Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor protocols, 2012(2), 199–204. doi:10.1101/pdb.ip067876
I’ve been thinking that a GC6 + red fluorescent protein vector would be great! Any idea when this will be available in the Penn catalog?
Thanks so much,
Kyle
Yes, Kyle: I dumped all 3 versions on PennU a while back. It should be online any time now.
Cheers,T
That’s great – thanks again!
thanks to ybot for sharing.
I contacted UPenn Vector Core and they told me that they do not plan to put these constructs into their catalogue, unless they get a few more requests. So, may be worth dropping them an email if you are interested.
Cheers,
Mike
mashby: I just checked with PennU just to make sure…
They confirmed that they currently produce the 3 syn-versions mentioned above for their catalog. They told me it will take ~3 more weeks until they are ready for ordering.
Further requests won’t hurt, though, and may speed up the process.
I still need to make sure that they concentrate the virus as much as possible – this tool needs high titer.
Cheers,
ybot
It looks like the 6s and 6f version have hit there catalog. They advised me that the 6m version should also be ready soon. Thanks again for the contribution.
Best,
Kyle
Finally. We were running quite dry ourselves …
I have to say that I know _nothing_ about this prep and cannot tell if the titers and viscosity are ok.
Good luck!
Hi everybody,
maybe some of you already noticed: PennU is holding back the current virus batch they produced. The reason is that I asked them to….
The titer of the two viruses was extremely low (~E12GC/ml >10-fold lower than the prep that I sent around to many labs for testing). Sadly, this is very unlikely to yield acceptable expression. Of course you could still try if you ask them to send the virus anyways.
They are currently repeating the rep – I cannot tell right now when they will be finished.
Best,T
The 6m version they just sent me is 7.18e12 GC/mL. I should know in about a week what the expression looks like. In my experience with other vectors, this has been sufficient – does this sounds like a workable titer?
Thanks,
Kyle
We have looked at the 6f version from UPenn (stated 9e12 GC/ml). 3 weeks post-injection, expression is widespread but fluorescence is pretty dim (compared to 6f alone). Will be interested to hear about Kyle’s results, and thoughts on whether upping the titre will help.
Cheers,
Mike
Hi Kyle/mashby,
this seems to me as if this would indeed be the 2nd PennU prep already (the first had even lower titers with 5.16e12 for 6s and 2.32e12 for 6f).
While the new titers for 6m/f are slightly higher, they are still not very good, sadly. They may have been ok for monocistronic expression – the bicistronic AAVs, however, are a little capricious…
As an example: the excellent PennU mRuby2-GC6s prep that we used over the last year had a titer of 3.7e13/ml. This yielded quite good expression in visual cortex – still, as desired, GC expression usually did not reach the level where it would fill the nucleus (~4 times lower expression than GC6s alone – rough estimate). That’s also the virus I sent out to many labs for testing.
We also have a PennU mRuby2-6F prep with a Titer of 1.53e13/ml. This is clearly usable but the expression level is noticeably below that of the 6S prep.
I also made a number of non–GeCI bicistronic AAVs. In all cases expression of the main construct (e.g. optogenetic actuators) was quite a bit below the level of their monocistronic counterparts at similar titers. Not sure why this is the case, but in my hands it’s reproducible.
So, yes: Higher titer will help a lot – the higher the better. It is rather unfortunate that the PennU preps again did not reach the >2e13 mark. I will ask them if they could post-hoc concentrate the virus.
Cheers,T
Thanks for that insight, Tobias. I can pretty much confirm the same pattern here with the 6m; the monocistronic vector produced higher levels of expression. Although, it had a higher titer as well (3.38e13 GC/mL) – so it’s hard to be certain what is the cause of the lower expression.
In any case, thanks again for sharing the vector – please keep us updated if any headway is made upping the titer.
Kyle
…we now have a few floxed versions on addgene as well:
https://www.addgene.org/Tobias_Bonhoeffer/
Cheers,
T
Very excited for the floxed version of the ruby-GCamP!
However, I was wondering if the virus has been tested in other brain areas rather then the cortex. I used it in the olfactory bulb, and unfortunately, I see some green cells (expressing GCamP) but not clearly expressing Ruby. I mean, the red fluorescence is very faint and diffuse. Do you have any explanation for this?
(I am using 2P excitation at 950 nm and a 610/75 filter)
Hi Martha,
sorry to hear it does not work well for you. My guess is that it’s the same as with pure GCaMP: some regions / layers just show more rapid nuclear fillling and ‘unhappy’ cells after GCaMP expression than others. If the cell is not healthy it is more likely to have a high resting Ca2+ level – which of course would lead to a far higher GCaMP signal and therefore increase the green/red ratio.
Maybe you can furhter dilute the virus to decrease expression?
There is also the possibility that there is some differential protein turnover that will change the initial stoichiometry of the red and green protein… but that’s handwaving.
Your filters and wavelength should be fine for coexcitation. Maybe check @1040nm to see if also the absolute red fluorescence is lower than in the rest of the cells.
Any chance we can see some side-by-side comparison (images, or tech specs) between GCAMP6 and GCAMP5, GCAMP3? And has Dr. Kim or other Janelians made UAS-GCAMP6 transgenic flies, and how do those compare to GCAMP3/5? Looking forward to reading the paper… any idea GCAMP6 will be published?
If I may:
Holy Crap – GC6s _is_ awesome!
did anybody tried it on Chlamydomonas reinhardtii?
Thanks
M.
Hi,
Is there a conditional (flexed) version of the Ruby.GCamp6s?
Thanks!
The UPenn vector core now provides Cre-dependent versions of our mRuby2-P2A-GCaMP6 AAVs (so far untested by us) :
AV-1-68720 AAV1.Syn.Flex.mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40(Addgene68720)
AV-1-68717 AAV1.CAG.Flex.mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40(Addgene68717)
AV-1-68719 AAV1.CAG.Flex.mRuby2.GSG.P2A.GCaMP6f.WPRE.SV40(Addgene68719)
[…] are a bunch of GCaMP6 mouse lines out now, from Hongkui Zeng (Ai93, Ai95, Ai96 are available), Cris Niell, GENIE (paper), […]