Welcome to the Tol2kit blog

This is a forum for discussing the Tol2kit; the Lawson lab's related system; and related topics.

The Tol2kit is a set of constructs that can be used with Invitrogen's multisite Gateway recombination-based cloning system to build expression constructs in a Tol2 transposon backbone (derived from constructs from the Kawakami lab).

At present each post serves as a topic under which visitors may post comments, either commenting or asking questions. Please post with your full name and institution (not just your first name or "Anonymous"), as it is helpful for other readers to know who is asking.

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Monday, January 29, 2007

Questions on the Tol2kit, 1/29-4/22/07

Ask general questions about the Tol2kit system by commenting on this post; we will do our best to answer in a timely manner. This posting covers questions from 1/29-4/22/07. Please add new questions under the appropriate later post.

29 comments:

jim lister said...

We have used the single Gateway approach, creating a couple of Destination vectors with GFP reporters for promoter analysis. Unfortunately because of the way these were made they won't be compatible with the Multi-site vectors the Chien and Lawson labs have made (i.e. they have R1/R2 sites instead of the R4/R1 combination) but they are compatible with the TOPO Entry vector pENTR/D-TOPO if you want to add attL sites to a promoter fragment via the vector rather than the PCR primers. (You still have to design a special 5' primer to do the directional TOPO cloning, but you may be able to use a 3' primer you already have.) pENTR/D-TOPO can also be used to make middle entry clones for the Multi-site system, I believe.

I can provide more information on these Destination vectors to anyone interested. I believe ours is the same approach described by Shannon Fisher except her vector has the minimal c-Fos promoter, for characterizing putative enhancers; our vectors are also in Tol2 but have no promoter (though one still has the rabbit beta-globin intron).

jim lister said...

I was wondering if (and how) the tranposase plasmid pCS2FA-transposase differs from pCS-TP? My lab has slightly modified pCS-TP to give it an optimized Kozak and I'm happy to make this available, but perhaps pCS2FA already accomplishes this.

-jim

Chi-Bin Chien said...

Hi Jim,

Thanks for the information about your destination vectors.

Looking at the sequence of pCS-TP, there were several features we didn't like: there were upstream mini-cistrons (start and stop codons), and no obvious polyadenylation signal; instead there was a polyA stretch, probably from the original cDNA.

pCS2FA-transposase has an optimized Kozak and the SV40 late polyA signal (from pCS2), and lacks the upstream start and stop codons. In principle it should be better, but we haven't actually compared it head-to-head with pCS-TP.

jim lister said...

Thanks Chi-Bin for posting sequences on the Wiki! I just wanted to make you aware of another potential option for plasmid distribution that I just discovered recently. The site URL is :
http://www.addgene.org/pgvec1

I've registered but not ordered or submitted any plasmids myself here yet. I think I still like/prefer the idea of ZIRC handling all this, but at the very least maybe there's something to be learned from the way this other operation works.
-jim

jim lister said...

I think it's important to point out the differences in the LR clonase enzyme mixes used for different Gateway procedures. I was about to try a multi-site reaction with some of the kit samples with some LR clonase II that we already had in the lab, but then I happened across a note on Nathan's site that one has to use LR clonase II *Plus* for a multi-site reaction to work.

Also, for anyone new to doing single Gateway, it took me a while to figure out that the difference between clonase and clonase II is just that the latter has buffer included in the mix with the enzyme. (I had clonase II but was working from a clonase I protocol, and spent an hour searching freezers for a non-existent tube of reaction buffer.)

-jim

Chi-Bin Chien said...

Thanks Jim for the suggestion about Addgene. We're aware of them, but when Nathan contacted them they were very particular about MTA issues (including for things like EGFP), and thus it seemed that there would be a lot of paperwork involved.

Chi-Bin Chien said...

Jim is completely right about the enzymes and we will clarify this on our wiki.

Arul said...

Hi, I am trying to use your p5E-MCS vector to clone a UTR element for shuttling into an appropriate expression destination vector. Since my insert was prepared by a conventional PCR without the use of att based primers, I thought of proceeding with restriction based method of cloning into the p5E-MCS vector. But I guess the CcdB gene is absent in this entry clone as its not shown in the sequence. So if I clone the insert by blunt end cloning protocol, I won't be able to screen for the positive clones as there appears to be no blue/white selection too. Could you provide me some suggestions in this regards. I could try to make fresh PCR products with att based primers.
Thank you
Arul

Chi-Bin Chien said...

Hi Arul,

You could indeed add att primers to your primers, as you suggest, and then do a BP reaction using pDONR P4-P1R.

Alternately, you could subclone conventionally into p5E-MCS. You are right, there is no gate (ccdB/cam) in those clone, and no way to do blue-white selection., If you're blunt cloning, you would just do it the old-fashioned way: phosphatase the p5E-MCS and do a parallel ligation without insert to measure your background. Should work fine though.

Chi-Bin

Arul said...

Hi Chi-Bin,
Thanks a lot for your suggestions. It really clarified the whole system. I decided to proceed with creating my own 5' entry clone by cloning in a pDONOR P4-P1r vector. Could you please let me know if the middle entry clone, pME-EGFP consists of GFP ORF with a minimal promoter. I just wanted to make sure with you.
THanks a lot
Arul

Chi-Bin Chien said...

Hi Arul,

No, the pME-EGFP clone is just an EGFP coding sequence, no minimal promoter. Nathan Lawson has made a clone with a minimal promoter, and this would be very useful to distribute.

best wishes,
Chi-Bin

Jim Amatruda said...

Hi Chi-Bin,
to follow up on the recent questions: I'd like to be able to analyze a promoter including parts of the first exon, so would like to be sure that I will fuse to a C-terminal GFP in-frame. Can I use p3E-EGFPpA for this? If so, do I need to generate a middle entry clone (containing the putative promoter) in a particular frame? Thanks
Jim

Chi-Bin Chien said...

Hi Jim,

If you're making the promoter clone by PCR, why not just cut off the first exon immediately before the start codon?

However, if you want to do what you said, there are two ways. 1) Use your promoter in a 5' clone, then pME-EGFP, then p3E-polyA; or 2) As you suggested, use p3E-EGFP-polyA, putting your promoter in the middle clone and using a filler (e.g. p5E-MCS) for the 5' clone.

Personally I prefer putting the promoter in the 5' clone as you will then have more flexibility for using for other things later on.

In either case, you do of course have to check the frame of the exon in the promoter clone. See the Invitrogen manual, which suggests a particular frame to use when designing att primers. We were careful to follow this with the p3E clones; I do not remember offhand whether we were careful with the reading frame at the 5' end of the pME clones.

jim lister said...

I suppose if one wanted to analyze a promoter fragment that included the first exon and part of the first intron as well, one would need a splice acceptor and maybe an IRES in the middle entry clone, right? Or is there another way to do this with the existing middle or 3' entry components?

Chi-Bin Chien said...

Jim (Lister), I think you're right. If your promoter fragment included a whole exon, you would need to add a splice acceptor in some configuration--with an in-frame GFP, or leading up to IRES-EGFP-polyA. There are certainly splice acceptor constructs out there, but we have not yet built anything like this.

jim lister said...

Regarding the Destination vector with the cmlc2:egfp transgenesis marker, has anyone established lines with this vector? I recently generated some lines using a similar strategy and many of the transgenic F1's have heart problems (but no other apparent defects), suggesting that sequences in the cmlc promoter may inappropriately enhance other nearby promoters (the other promoter I was using was hsp70, and I was very careful not to heat shock the embryos). I haven't yet checked for inappropriate expression of my non-marker transgene in the heart, but if someone could tell me they've NOT seen this problem with pDestTol2CG2 or the like I'd be grateful.

By the way, I saw this in multiple founders, but all had been injected with the same construct. It was also Tol2-derived so should have given single-copy insertions. I was only using about 250bp of the cmlc2 promoter versus 900 that are in pDestTol2CG2, although in my plasmid I had the cmlc cassette in the same orientation as my other, non-marker transgene (i.e. head-to-tail, rather than tail-to-tail) which places it closer to the hsp70 promoter than it would be in pDestTol2CG2.
- Jim

Adam Rodaway said...

Hi Chi Bin, I've made a DONR vector with P4P3 sites flanking the selection cassette. This should allow people with pre-existing promoter-reporter constructs to clone them intact into your DEST vectors without using multisite.
If you like, I could drop it in the post to you so you could include it with your distribution.

Kristen Kwan said...

Adam -- your DONR vector sounds really useful! We would be grateful to have some. At the same time, I think we are working out a way for distribution to be handled by ZIRC in the future -- we'll keep you apprised of our progress. Thanks!
Kristen

Kristen Kwan said...

Jim -- we in the Chien lab have started establishing lines with pDestTol2CG (specifically Melissa Hardy). She has isolated founders for three different lines (all with the hsp70 promoter). Melissa has not noticed obvious heart defects in the F1s yet; however, her oldest clutch is about 1 month old. When did the heart problems become apparent?
-- Kristen

jim lister said...

Hi Kristen,

Thanks for sharing those results. In some of the embryos from my founders the defects were very early, e.g. the hearts didn't loop correctly. Some looked pretty good for a couple of weeks, but I haven't had any survive longer than a month yet. I do however have founders from another construct that includes the same cmlc cassette, and at least some of the F1's look fine at almost two months. I suspect it has a lot to do with what the ORF is that is being expressed: if it's something that even at low levels could perturb heart development (which I suspect would be a very long list), people might want to be a little careful.

I will probably still try using pDestTol2CG2 to make the same transgenic, and post the results here.

-Jim

Anonymous said...

Hi,
I am trying to amplify a promoter region to put in the 5' entry vector and am having real trouble getting it to amplify. It is very AT rich, unfortunately. I was wondering if you have any suggestions for PCR mix and annealing temps that would better allow those long overhangs to bind and extend.

Thanks,
Corey Snelson

Arul said...

Hi Chi-bin, Thank you for clarifying the promoter issue of pME-EGFP. I believe that the same is true with pME-nlsEGFP too. Since, I am planning to screen for a particular enhancer element. I guess I should first create a middle entry clone with a minimal promoter fused to EGFP and then go ahead with the multisite cloning of my putative enhancer p5E clones. I hope I am on the right track.
I do realize that you have created this tol2 destination vector for multisite cloning purpose. If I need to clone a single construct into a tol2 destination vector, I guess I would need a tol2pDEST with R1R2 sites. I wanted to know if such a vector has been designed...
Thank you
Best wishes
Arul

Chi-Bin Chien said...

Hi Arul,

Yes, Nathan Lawson's lab has made a Tol2 destination vector using R1-R2 sites. See their webpage at:
http://lawsonlab.umassmed.edu/gateway.html
and look in the table of destination vectors for pTolDest.

Chi-Bin Chien said...

In response to Corey Snelson's comment: we have had good success with long genomic PCRs using Takara LA Taq (available in the US through Millipore), as suggested in Shannon Fisher et al.'s Nature Protocols paper (2006), vol 1:1297.

Ted Allison said...

I am trying to acquire a copy of the Invitrogen multisite Gateway manual 11/29/04. The links I can find most easily on your wiki & blog seem to all point to the new version of the manual, Version D from 7/March/2007. Can you please suggest a way to get the older version?
Thanks for the helpful site and plasmids -
Ted Allison, U Michigan

Chi-Bin Chien said...

Ted--Indeed, Invitrogen has posted an updated version of the manual, which I had not seen until now. It looks like it should be fine for your purposes, I believe it has just been adjusted to reflect use of the LR Clonase II Plus enzyme. However, I still have a PDF of the old manual and can send it to you if you email me directly.

Claudia Gerri said...

Dear All,
I'm a PhD student in Stainier Lab.
We just received an update list of plasmids, with a sheet that describes those clones. They said: "complete documentation with maps and sequences will be available soon on the Tol2kit wiki".. But actually, I didn't find it.
I'm interested in pDestTol2pA, especially in pDestTol2pA3. Does someone know where I can find map and sequence of it?

Thanks.
I'm looking forward to your reply.
Best regards,
Claudia Gerri.

Kristen Kwan said...

Dear Claudia,

We appreciate your patience. We are in the process of updating the wiki site (which needs to be migrated to a new server and the software updated significantly) before the sequence information is posted. In the meantime, please email me (kristen.kwan@genetics.utah.edu) and I will email you whatever sequence files you need.

Also, if possible, in the future please post your questions and comments under the most recent post here on the blog - it will make it easier for us to find your questions! (This post was closed in 2007.)

Best,
Kristen

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