Our lab is looking to use CRISPR to tag a histone (his-72) with mTurquoise2. Has anyone used mTurquoise2 in the worm yet? Specifically the germline? Has anyone used it as a histone tag? We have the Goldstein lab worm codon-optimized vector (pDD377), but haven’t seen any publications with mTurquoise2 in the worm. Any info would be appreciated.
We were going to do the same thing and send to the CGC, but I haven’t talked anybody in the lab into doing it. We were also going to tag a plasma membrane protein with mTurq2, maybe RAP-1.
It should work. mNG::HIS-72 is super bright, so mTurq2 should be as well. Do use Dan’s codon-optimized mTurq2 and you should be fine. There is some chance of germline silencing. We do not have a germline optimized (GLO) mTurq2, but my bet is that multiple tags will get you what you want. Or, remake GLO yourself.
We’ve gotten all the reagents for the histone marker and are going to inject this week. If you’d like us to send you the strain, just shoot me a message!
One suggestion would be that if the strain works as expected, to disseminate the result by Micropublication. https://www.micropublication.org
It’s a new publication platform being developed by Paul Sternberg, Tim Schedl, Daniela Raciti, Karen Yook, and Todd Harris. It’s a platform to publish small pieces of data (gene expression, methods, integrations, phenotype, etc.) which will be peer reviewed and rapidly published online with a digital object identifier, so they are citable. It’s got a lot of potential to get data, such as the strain you’re describing out to the community and to get your reagent/data found and cited.
Great suggestion Jordan! Some of these strains should be obvious and needed by many labs, this is an excellent way to avoid having multiple labs make the same line.
Sorry, I was slow to see this but have something to add. It sounds like we’re all after the same thing - a strain with blue histones in the germline. Here’s what I’ve tried: Both htz-1 and his-74 are most prominently expressed in the germline, so I created mTur2-SEC plasmids to tag the endogenous genes and injected each multiple times. I could get hets and a lot of bright blue and very dead embryos. I then created mTur2-SEC plasmids with a bicistronic spacer to drive expression of his-58::mTur2 with the glh-1 promoter thinking that H2B-GFP transgenes seem to not be toxic and we’ve used this glh-1 bicistronic approach to drive other things specifically in the germline. (note that the rla-1 bicistronic spacer does cause some misexpression in the posterior pharynx.) I wondered then if it was an mTur2 problem, so I swapped this out with mTagBFP2. Same thing. My conclusion is that tagging endogenous germline-expressed histones using CRISPR is toxic, while our old transgenic histone reporters are expressed at lower levels and selected for viability. After several injections I gave up, but if someone wants these histone SEC plasmids to try their own injections I’m more than willing to send what I have.
My only comment there is that we know that CRISPR tagging of HIS-72 works and is strong and gorgeous with mNG (see Dickinson, 2015). My lab uses this mNG::HIS-72, no problems. Since we know it knocks in efficiently,it is a great bet to reproduce with some blue fluorescent protein.
Just checking: did you include a good linker in your tags that caused problems? We accidentally left a linker out of one CRISPR of an essential gene, and it caused a lot of problems. Prior tagging of the same gene using the same site was tolerated, but in that strategy we had a linker.
We have been using SAGGSAGG, after the many FRET constructs made by Matsuda. But others use SG(n) to good effect.
Ours have the GASGAS linker in the SEC vectors. At the time I had some evidence that htz-1 and his-74 were going to be more germline specific and expressed in both oocyte and sperm, so I didn’t try his-72. It would be a simple thing to swap in his-72 into our BLUE-SEC constructs and try it again, but some activation energy may be required after getting burned last time
We finally got some blue worms! We’ll be back-crossing, analyzing, and freezing them over the next couple of weeks. The first image is of the germline, and the second is of a two and four cell embryo in utero.
Our lab doesn’t have easy access to a flourescent stereoscope with the right filter set, so I don’t know about that. We used a 450/50 emission filter with maximum blocking at 405 nm, and maximum intensity from 425-475 nm.
UPDATE: Dillon in Joshua Bembanek’s lab generously sent us these worms. We were able to see them on our stereofluorescence scope with CFP filters. My lab is eagerly crossing these into some of our red or green cell bio markers to visualize with a nice live blue nuclear control! Hat tip to Dillon and Josh!
