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.

To help prevent spam, word verification is turned on: you will have to do a "word recognition" task before you can post a comment.

Saturday, April 26, 2008

Questions on the Tol2kit, 4/26/08-6/8/09

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 4/26/08-6/8/09.

78 comments:

Kristin A said...

Hi, does anyone know how big DNA inserts can be to successfully recombine? We are specifically interested in the heat shock and vectors for standard transgenesis.
Thanks.

Chi-Bin Chien said...

Kristin--

In terms of entry clones, we usually put the biggest fragment (enhancer/promoters) in the 5' clone. We have fairly often used 8-9kb fragments here for BP reactions. For middle clones, we have several 4-5kb fragments that have worked for BPs.

As for LR reactions, our record at present is a 14 kb 5' clone plus a 1 kb middle clone plus a 2 kb 3' clone (all insert sizes), into pDestTol2CG2. Total size about 23 kb. For these really big constructs, we use electrocompetent CopyCutter bacteria from Epicentre.

If you just want to make hsp70:gene-of-interest, you should have no problem at all.

Anonymous said...

Hi,

I am curious to learn about people's experiences using the viral 2A peptide for co-expressing proteins in zfish. The Provost et al. 2007 publication in Genesis suggests that it works well. Has anyone else used this strategy successfully?

Thanks,
Geoff Burns

Chi-Bin Chien said...

Hi Geoff,

Yup, the 2A works well in our hands. We've made some 3' clones that should make their way into future versions of the Tol2kit. The only caveat is that you end up putting a ~25 aa C-terminal fusion on the first protein (from the att site and the 2A), and a proline residue fused to the N-terminal of the second.

Kazuki said...

Hi, I have a general quetion.
Does the Tol2 kit contain donor vectors (especially pDONRP4-P1R,pDONRP2R-P3)?
Or we have to buy them from Invitrogen?
I think the MultiSite Gateway Three-fragment Vector Construction Kit is too expensive...

Kazuki Sakai

Tom Hall, Currie Lab said...

Hi, I want to do in vitro transcription of RNA. Presumably I make a construct consisting of p5E-MCS, pME-my gene, p3E-pA. Then use T7 to transcribe. Is this the most sensible approach?

cheers,

Tom Hall
Currie Lab

jim lister said...

Hi Tom,

You could also use SP6 polymerase with an expression clone made with p5E-CMV/SP6. Looks like your plan with T7 should work though, and at least under some conditions T7 is the more robust enzyme.

Also, to answer Kazuki's question, the Tol2 kit upgrade I just received (thanks Chi-Bin and Chien lab!) includes those donor vectors.

-Jim

Tom Hall, Currie Lab said...

Hello again, what are the relative advantages of using H2A-mcherry versus nlsmcherry? Have you also made, or would it be worth making H2A-EGFP?

cheers,

Tom

Chi-Bin Chien said...

Hi Tom,

The nls fusions tend to leak a bit into the cytoplasm. The histone fusions are cleaner during interphase; furthermore, during mitosis they label the chromosomes, allowing visualization of mitotic figures.

We've made H2A-GFP fusions and can include them in a future distribution.

Chi-Bin

Erica said...

A colleague of ours that we distributed clones to has sequenced plasmid #396 (pCS2FA-Tpase) using Sp6(F) and T7(R) and sent me the sequence.

We noticed that there is extra(neous?) sequence 5' and 3' of the "transposase" gene annotated in the wiki sequence that does not align to any other part of the vector. I blasted and they appear to be noncoding seq from the annotated tpase DNA D84375 directly up- and downstream of ORFs 1 and 4, respectively.

Maybe we have an earlier version of 396? Otherwise the wiki seq may need to be updated? We've used RNA made using Sp6/not1 and appears to be working-good efficiency of TG for us, but please let me know if you think we should acquire another vector without this extra sequence. I'd also be happy to send their sequence along.

Thanks!
Erica

Anonymous said...

I have a maybe already asked question about the pME-mCherryCAAX clone (clone 232) that was distributed in the kits bevor 2007.
Does this H80D exchange has a effect on the proper function of mCherry fluorescence?

thank you for your answer,
Jochen

Chi-Bin Chien said...

Erica-

The transposase clone that you describe sounds like the original clone from Kawakami lab, which presumably has gotten mixed up with the pCS2FA-transposase clone from our lab. We hope that this mixup did not happen at our end! and are resequencing the latest maxiprep to confirm.

Chi-Bin Chien said...

Jochen--

We have not done a head-to-head test ourselves, but have heard several anecdotal reports that the H80D mCherry is indeed much dimmer than the original.

Chi-Bin Chien said...

Erica-
We have resequenced the pCS2FA-transposase maxiprep that we distributed, and it is correct. So it sounds like someone at your end has mixed it up with the original Kawakami transposase clone.

As you suggest, that clone should also work, though potentially the RNA will not be as stable as a pCS2-derived one, so you may have inject more RNA for the same effect.

Anonymous said...

Hi,

We've made a couple of transgenics with the b-actin promoter from the Tol2 kit. The expression from this promoter in the progeny of the injected fish is surprisingly variegated. Has anyone had a similar experience with this promoter?

Thanks,

Howard Sirotkin

Chi-Bin Chien said...

Howard--

We have not noticed variegation with the bactin2 transgenics that we have made. Of course different constructs may show different sensitivity to position effects.

Do you know for sure whether these transgenes are transposon insertions, or might they be transposon-independent DNA insertions? These would likely be rearranged concatamers, and might be more prone to position effect variegation.

Jonathan Rosen said...

I am having a problem entering a tol2 destination vector into the computer program Vector NTI. When I copy in the sequence for pDestTol2pA2, Vector NTI sees this as an entry vector, not a destination vector, although it recognizes the R4 and R3 sites. I would be grateful if you had any suggestions on how to fix this problem.

Chi-Bin Chien said...

Jonathan--

We do not use Vector NTI, so I don't know for sure. However, if I had to guess, maybe the new Vector NTI program is set for Invitrogen's new "Multisite Pro" system (up to four entry clones into a destination vector with attR1-R2 sites).

Try poking around and see if there's a setting to tell NTI that it's just "Multisite", not Multisite Pro.

Jonathan Rosen said...

Thank you for your response! I figured out the problem, which I will explain here in case anyone else has the same issue. Vector NTI wasn't recognizing the ccdB sequence in the vector, so I had to put it in manually by going to Feature Map --> add. After this, Vector NTI treated it as a destination vector.

Rachel said...

Hi there,

I will be using the pDestTol2pA construct and I want to place a promoter between the Tol2 site and the attR1 site. I'm having a hard time figuring out where this is located on the construct sequence given on the Chien website, but I'd like to know if there are any restriction sites that I could use to clone in my promoter.

Thanks.

Chi-Bin Chien said...

Rachel--

The destinatino vector pDestTol2pA (and pDestTol2pA2) does *not* have an attR1 site, only attR4 and attR3 sites. Can you clarify your question?

PS--Please post with your full name...

Anonymous said...

Hi there,

I would like to clone in a promoter between the first tol2 site and the attR3 site so that when I flip my insert in between the attR3 and attR4 it will be under my own promoter. My question is whether there are any restiction sites between the first Tol2 site and the attR3 site that I can use to clone in my promoter.

Thanks,

Rachel Monpetit

Anonymous said...

Hi again,

Sorry I want to clone in my promoter between the first Tol2 site and attR4.

Thanks,

Rachel Monpetit

Chi-Bin Chien said...

Rachel--

I'm confused: why not use the destination vector in the way it was intended? A three-insert multisite Gateway reaction will let you put your promoter in the 5' clone, a coding sequence in the middle clone, and a polyA or 3' tag in the 3' clone.

If you really need to clone in conventionally between the Tol2 end and the attR4, you can figure it out from the Genbank file available on the wiki. The Tol2 exon4 fragment is dispensible, as is the M13R sequence. ApaI and NsiI lie within this region and are unique in pDestTol2CG2 (and pDestTol2pA2).

PS--Have you read the Tol2kit paper? It might help...

Anonymous said...

Hi,

I was wondering if there had been any experience with transgenic markers getting silenced. For example, have you noticed using the vector 395, the backbone with the cmlc:egfp marker, that the gfp gets silenced when paired with certain promoters that had been gatewayed in? There had been some rumors of this awhile back.

Thanks, Colleen

Amayra said...

Dear Chi-bin Chien,
I think I posted my comment in the wrong date I send it again in case.

First, I also wanted to thanks all the people involved in developing and sharing this system and constructs.It is great!. Thank you; Chi-bin Chien, Kristen Kwan,Ben Mangum and Esther Fujimoto... also for making a really useful web and blog. Also to the people that did the apE programm.

I have some problem with the IRES-caaxEGFP. I made a construct carring the pt2(1.5hspGal4VP16IREScaaxEGFPpA) using the the 5',ME and 3'entry clones that are in the tol2 kit and the pDestTol2pA2(394) also from the kit. I checked the sequence and seem to be all right however when I injected this construct to make and small enhancer trap, from the mosaic EGFP positive embryos, quite a lot have strong EGFP in the yolk from the end of gastrulation on wards. The yolk starts getting black (cell death?) and by 48-72hpf lot of them dye. Did you find any similar thing using the caxx-EGFP?.I have try different concentrations and seem to happen the same. The strange thing is that the injected embryos that do not express EGFP in the yolk survive well. Before I injected embryos with the same concentration of pT2600bphspGal4VP16pA (without any Ires memebrane EGFP)and I did not have this problem. To my knowledge no one has reported leaking or basal expression of 1.5hsp in the yolk. Is that right? Did you try any minimal promotor (not the c-fos) that works well(not basal expression)in zf?.
Thanks again for all your help.

Amayra

Gray said...

I'm looking for advice as to which clones to choose to make:

5' entry: 3.5kb promoter for GOI
Mid entry: GFP w/ stop
3' entry: <2kb enhancer for GOI

Any advice? Thanks in advance.

Chi-Bin Chien said...

Amayra--

Your results are a bit puzzling. We have not noticed toxicity with the IRES-EGFP-CAAX in other contexts. The hsp70 promoter should not be leaky in the yolk. HOWEVER, many GFP constructs will express in the yolk if you inject DNA into the yolk rather than the cell at the 1-cell stage. Where are you targeting your injections?

Chi-Bin Chien said...

Gray--

I'm afraid I don't understand your post. What is "GOI"? What choice are you asking about?

Note that pME-EGFP-stop is already included in the standard Tol2kit.

Amayra said...

Dear Chi-bin Chien,

Thanks for your reply. I try to inject the embryos before one cell stage in the yolk free region of the embryo. I injected the mix with phenol red so normally I am able to see the drop in this area. What you recommend me to do? inject later maybe?. I do not understand why a EGFP construct that does not have a promoter (just minimal promoter like hsp) is expressed in the yolk (w/o finding and enhancer).
Thanks for your help.
Amayra

Caroline Parkin said...

Hi,
I no longer have access to Lasergene and I'm trying to get to grips with Ape. I'm trying to add your sequences for the tol2 kit components whilst retaining the features that you've annotated, but I can't seem to manage it.
Please could you tell me how to add the sequences?
Many thanks,
Caroline Parkin

Chi-Bin Chien said...

Caroline--
If I understand your question properly, you would like to open the sequence files in ApE with all the components annotated. The easiest way is to not use the FASTA files from the Tol2kit wiki, but instead, the annotated Genbank-format files. ApE can read the Genbank files directly. All of the features should be highlighted in the sequence, and show up on graphic maps.

Margaret said...

I've already asked a couple of you this privately, but on Chi-Bin's suggestion I'll ask the whole community. We've been using pDestTol2CG2 as our primary destination vector, and we're finding a lot of germline carriers of the cmlc2:EGFP, but not of the promoter:gene combinations we put into the destination vector. It's unclear whether the other portion is getting silenced or is somehow not integrating. Has anyone else seen this? Might it just be a matter of position effects? And perhaps most important (practically speaking): How many potential founders do folks screen to get strongly expressing lines?

Thanks,

Margaret Mills
Parichy Lab
University of Washington

xiufang said...

Dear all:
I am doing with Tol2 kit to make expression plasmid but I confused with the choice that whether the entry clones need to be linearized or not before LR reaction. somebody said the linearized clones can increase the recombination efficiency, does anybody have this experience about it?
In addition, if I want to establish a stale transgenic zebrafish line with the Tol2 transposon method, do I need to linearize the expression plasmid before injection? what is the difference between the linearized contructs and the circular ones for the germline integration rate?
thanks a lot!
xiufang pan
11-14-2008

Chi-Bin Chien said...

Xiufang--

It's not necessary to linearize entry clones before LR reactions (though in principle this could reduce background). We routinely do LRs with circular substrates and have >50% of clones correct.

It is also not necessary to linearize the Tol2-containing DNA plasmid before coinjecting with Tol2 transposase RNA. I don't know of any cases where linear vs. circular plasmids have been tested for integration rates.

Jason said...

Last December, Jim Lister noted that you were making some entry vectors with Cre.

Any luck with these?

I am interested in making a GFP-v2A-Cre construct for testing and then make a mouse knockin of Exon-v2A-Cre.

Thanks, Jason Stoller

jim lister said...

Hi Jason,

I've made but not tested a middle entry clone with the ERT2-Cre-ERT2 fusion described in Matsuda and Cepko 2007 PNAS 104:1027. I'm happy to share it if you're interested in trying it out -- email me at jalister@vcu.edu.

Cheers,
Jim

Tim said...

Hello all,

For some experiments, I would like to combine an enhancer + promoter + gene + polyA (4 elements). As I see it, the options are

1. combine certain elements (e.g. make a middle entry clone with a minimal promoter + eGFP, and make the enhancer the 5' clone. The problem with this is that if I then want to drive a different gene for another experiment, I have to create a new middle clone each time).

2. Clone the enhancer upstream of the attR4 site in the destination vector. This abandons the convenience of gateway cloning, but allows for mixing and matching different combinations of enhancer + 5' clone + middle clone + 3' clone.

3. Convert to a "multisite gateway pro" system for up to 4 sites. The Tol2kit is not compatible with the 4-site "pro" system, but maybe it could be extended easily without having to redo all the existing clones. If I convert the attR4 site in the destination vector to an attR5 site, and create a donor vector with attP5 and attP4r sites, then that would allow for a 4th site upstream of the other three. It wouldn't be the same site order that the standard multisite gateway pro system uses, but I don't see why it wouldn't work (unless an attP5-attP4r vector self-recombines too much?).

Have people tried any of these strategies? Does anyone have other suggestions? Are there plans to expand Tol2kit to a 4-site system?

Thanks for your advice,
Tim Howes

jim lister said...

Hi Tim,

A variation/alternative to your option 2 would be to use Clontech's In-Fusion system (see their website) to clone your enhancer upstream of the attR4 site. We are just starting to play around with In-Fusion -- perhaps others out there have more experience with it and can comment if it would be appropriate to use in this context? All you basically need is a unique restriction site upstream of attR4 (pDestTol2pA2 has a unique XhoI site but I don't see anything for pDestTol2CG2). If you were to replace the XhoI site with a much rarer cutter you would have an easier time if you wanted to introduce enhancers after first doing the LR Gateway reaction to get everything else in place (i.e., less likelihood that your Gateway stuff would introduce another of the same site.)

Cheers,
Jim

Chi-Bin Chien said...

Tim--
We at least have no plans for switching to the Multisite Pro 4-insert system--that would be a lot of work!

The way that we have solved the problem of adding a basal promoter is to make p5E-FA-bas, a 5' entry clone that ends with a basal promoter, and has upstream eight-cutter sites (FseI-AscI). Thus you can PCR up your enhancer with Fse/Asc ends, and use conventional ligation to build your 5' clone. Then you have:

(enhancer-bas promoter)-(gene of interest)-(3' tag).

Note that if you only need a pA signal as the 3' tag, you could also use the one in the pDestTol2pA2 and pDestTol2CG2 backbones. Then you could put the enhancer in the 5' clone, basal promoter in the middle clone, and gene of interest in the 3' clone.

Jon Hildahl said...

I'm looking at setting up multiple promotor constructs with gfp or mcheery and a Tol2 backbone. I was thinking an efficient way of doing this would be to generate one construct and then change the promotor with PCR products containing attP sites, so in essence using the construct as a Donor vector. My question is if it's possible to incorporate attP sites in PCR primers?

Chi-Bin Chien said...

Jon--

attP sites are quite long (>100 bp I think) and so not practical to include in PCR primers.

If you are only planning to try a handful of promoters, it is probably easiest to just do multisite LR reactions for each one. These are really quite efficient.

If you plan to do large numbers of promoters, it is in principle possible to take an expression clone, e.g.

pDestTol2pA2; beta-actin:EGFP-pA

and do a "reverse BP" reaction using pDONR P4p1R to replace the beta-actin promoter with an attR4-R1 cassette:

pDestTol2pA2; attR4-R1-EGFP-pA

You could then just do single recombinations using attL4-L1 entry vectors to make the new expression clones that you want to.

There are published methods for the reverse BP; the principle is that you do a BP reaction, but select using ccdB-resistant bugs and chlor/amp, rather than kan as you would for a normal BP reaction (I think I have this right).

Chi-Bin Chien said...

Colleen and Margaret--

Sorry for the slow reply; I somehow missed Colleen's original post. Re the issue of whether green hearts always correspond with expression of the main transgene when using pDestTol2CG2:

1) It is important to distinguish between integration and expression. For bona fide transposon insertions, the main transgene should always be integrated whenever the cmlc2:gfp marker transgene is (since they are on the same piece of DNA). Remember though that some fraction of integrations will be transposase-independent integrations of bare plasmid; these are likely to be rearranged concatamers in which the marker transgene could potentially have been sheared away from the main transgene.

2) That said, expression and integration need not correspond. There are at least two possible reasons:

a) Position effect. The location of integration could affect the main and marker transgenes differentially. This is especially true since the cmlc2 promoter seems very strong. If your main transgene's promoter is weaker, perhaps it gets silenced but cmlc2 does not.

b) Interference from cmlc2. It is also possible that for certain constructs, or in certain integration positions, the cmlc2 promoter silences the main transgene. In our published paper, we did experiments to show that the cmlc2 promoter does not interfere with the hsp70l promoter. However, the heatshock promoter is very strong, and this result could certainly be different for a weaker promoter. We have heard anecdotal reports from other labs that weak promoters can indeed be silenced by the neighboring cmlc2 promoter.

Raquel Castellanos said...

(Sorry if this comes up twice but I originally posted this in the section for questions posted before April 2008.)

Hi everyone,
I am setting up an enhancer screen, testing upstream conserved regions of my gene, and putting these into the 5' Entry Clone. Since the promoter for my gene isn't defined, I decided to use the hsp70 promoter included in the kit and recombined it into the middle entry clone, and also recombined mCherry into the 3'entry clone. I figured the promoter would work specifically under the influence of the enhancer, without being heat shocked. The problem is that at this point I have tested 3 conserved regions but they all give me the same expression pattern of mCherry (which by the way is very strong). I have tested a control construct Hsp70-mcherry-polyA, but it gives me the same pattern without heat shocking! I am very perplexed. The regions where I see fluorescence are the muscle in its body and the heart. Has anyone else had this problem, or have any advice? I would really appreciate any help! Thanks in advance!

Chi-Bin Chien said...

Raquel--

The hsp70l promoter is known to drive some constitutive expression, e.g. in the lens after 48 hpf and also at low levels in muscles. I'm surprised at how strong you say the muscle expression is, but am not surprised that it's there. The groups that have enhancer trapped using hsp70l as a "minimal" promoter have found that the majority of their lines express in muscle.

A better choice would be the basal promoter-EGFP middle clone that Nathan Lawson's lab built. This is basically just a viral TATA box plus a little bit of 5' UTR from an actin (?) gene, hooked up in front of EGFP. See the Lawson lab website for more info.

Jon Hildahl said...

Hi,

I'm wondering why the pENTRDsRedEx2 vector from the Tol2kit uses DsRed instead of mCherry? Do you know if this is the monomer or tetramer form of DsRed and what are the best filters for detection of mCherry and DsRedEx?

Thanks,
Jon

Chi-Bin Chien said...

Jon--

pENTRDsRedEx2 is not part of the Tol2kit; I think it's one of the Lawson lab clones. Likely it was made before they started using mCherry, but for all I know DsRedEx2 is brighter than mCherry. The mCherry spectra are in the original publication (Shaner et al., 2004). I imagine the DsRedEx2 spectra are either published, or available on a company website.

gerosenf said...

Hi, are there any other transgenesis markers available for the tol2 kit DEST vector such as crystallin-EGFP, which marks only the lens of the eyes?
Thanks,
Gabriel Rosenfeld
Evans Laboratory

Anonymous said...

Hello,
Recently our lab has been injecting Gateway constructs with and without Transposase and are not seeing a difference in transgenesis between +/- Transposase groups. We have checked the integrity of our RNA via gel analysis and it's fine. Has anyone else seen this result?
Thanks much,
Emma Oster, Novartis Institutes for BioMedical Research

Chi-Bin Chien said...

Gabriel-

We have not made transgenesis markers other than cmlc:egfp, though others may have.

Chi-Bin Chien said...

Emma--

We routinely see significantly more expressing cells when we coinject Tol2 expression constructs with (rather than without) transposase RNA. Perhaps you have RNAse in your injection solutions?

Have you tried a control expression construct, like beta-actin:GFP-pA?

Jon said...

Hi,

I'm having trouble with LR reactions for multiple genes. The BP reactions work well, but when I try 3-way LR reactions using LR clonase II plus and the pDest Tol2pA destination vector (obtained from the Lawson lab, I use equamolar concentration of dest and entry clones)the reaction doesn't work. I get good growth on the amp plates with 100+ colonies, both clear and opaque. I usual prepare mini-preps of 4 clear colonies and then perform restriction enzyme digestion and PCR to confirm the constructs. It appears that the clear colonies contain the self-recombined destination vector. Have you heard of this being a problem and do you know of any remedies?

I've also be having trouble with the pDONRP4-P1r entry clone self-recombining during BP reactions. I tried the selection protocol available at the Lawson lab webpage, but I haven't been able to isolate any colonies that don't self-recombine. Do you know of any alternative means to avoid self-recombination with this vector.

Thanks

Chi-Bin Chien said...

Jon--sounds to me like your preps of destination and donor vector are bad (i.e., have picked up disabling mutations). pDONR P4-P1R is notoriously difficult to grow up. pDestTol2pA2 is easier, but we have occasionally had trouble (sequencing usually reveals a mutation in the ccdB gene). I recommend getting proven aliquots of both and starting over with growing them up. We have detailed instructions on the Tol2 wiki.

Since you didn't post your full name or address, I don't know where you are; there may be someone else nearby to get aliquots from.

Jon Hildahl said...

Sorry, my full information is Jon Hildahl at the Alestrom zebrafish lab/Norwegian School of Veterinary Science, Ullevålsveien 72, PO Box 8146 Dep, 0033 Oslo, Norway. I'll check around to see if I can find a new clone, but if you know of anyone in the area I'd be greatly interested. Thanks again for the help with this excellent blog.

/Jon

greenfish said...

Hello, Prof Chien, does the most recent vision of Tol2 kit include the 3' entry vector p3E-IRES-RFP(or dsRed)?

Does any one have this plasmid, and is willing to gift some to us?

Zhiqiang Zeng, Edinburgh Cancer Research Centre (zq.zeng@ed.ac.uk)

Anonymous said...

Hi,

We are trying to make a handful of different BAC transgenics and wanted to learn if anyone had tried putting ISceI or Tol2 sites around a BAC insert and then tested for increased germ-line transmission frequency.

Thanks,
Geoff Burns
MGH, Boston, MA

Chi-Bin Chien said...

Zhiqiang--

All of the current Tol2kit clones are listed on the wiki. We have thought about including IRES-RFP clones in future releases. IRES-mCherry is likely to be quite dim.

Chi-Bin

Chi-Bin Chien said...

Geoff--

Indeed, Koichi Kawakami has placed Tol2 ends into a BAC using bacterial homologous recombination and used this construct for transgenesis. I believe that he presented this work at the recent Asilomar PI meeting.

Anonymous said...

Chi Bin,

Thanks for the information. Do you recall what percentage of potential founders sent the BAC germ-line?

Cheers,
Geoff

Chi-Bin Chien said...

Geoff--

Sorry, I wasn't at the meeting and so did not hear the talk. The other potential advantage, besides possibly improved integration rates, is that you should presumably get clean single-copy BAC integrations, which would be easy to map by inverse or LM-PCR.

Brian Clark - Link Lab said...

Has anybody made entry clones containing destabilized GFP or RFP/mCherry? We are looking to test the activity of promoters driving Gal4, so ideally we'd like UAS:destabilized Fluor construct.

Chi-Bin Chien said...

Brian-
That would be useful, but we don't have such clones. I can probably find a clone containing destabilized GFP for you, if you'd like to make a middle clone yourself.

Chris S. said...

Has anyone generated a p3E-IRES-H2A-EGFPpA that they wouldn't mind sharing. Thanks, Chris Sansam (sansamcl@mit.edu)

Arul said...

Hi,
I received the pDONRP4-P1R vector with the kit. When I transformed the vector on ccdB tolerant cells, I obtained two kinds of colonies on the plate. A predominant population of small translucent colonies and a minor number of opaque and large colonies. I first thought that the ccdB competent cells were contaminated. I transformed the other donor vectors and I got uniform kind of colonies. I performed the self complementation screen of Lawson lab on the P4-P1R vector. I picked up 8 colonies and none of them seem to be the right ones. Could you please help me?
Thank you
Best wishes
Arul

Emilie M. said...

Hi,

I have been trying to recombine three different PCR products in pDONRP4-P1r. After the BP reaction and transfo of bacteries that are not tolerant to ccdb, we have thousands of colonies, but none of them contains the insert... since we are sure the vector we use contains the cassette, it seems that we have a intramolecular recombination (this phenomen occurs even when we do not pu any insert in it, just enzyme)... Did it already happen to you? what can be the solution to promote the insertion of my product?

thanks a lot,

Emilie.

Clara said...

There seems to be a lack of congruence between the attR1 sequence and the p5_MCS whole sequence. While attR1 appears as ...ctatgaaTcaactactta..., p5_MCS shows the following sequence ...ctatgaaCcaactacttag...

Probably the mistake is in the vector whole sequence, I am right?

Qing said...

Hi, I want to make put my promoter of different lengthes in the 5'entry clone and make Promoter-GFP constructs. Do I have to consider the reading frame shift problem? What if I put the promoter-exon1-intron1-part of exon2 in the 5'entry clone?

Which is the most commonly used Taq? Will the Taq introduce an additional A into the PCR product and will this influence the multisite recombination and reading frame?

Thanks a lot!

Flip said...

Hi,
I am trying to make a very large construct and I am not being able to.

Briefly I do a typical 10um Lr reaction O.N. and then I clean the reaction with a Quiagen reaction cleaning kit(PCR kit), I resusped in 10ul and I use 5ul for transformation.

I am using electrocompetent CopyCutter bacteria from Epicentre for that porpuse but something is going wrong as I do not get any colony.

I wonder if anyone has any protocol that could share with me.

Thank you very much for your help

Filipe Sousa

Molly Nyholm, Grinblat Lab said...

Hi,
Does anyone use vector NTI to make their gateway plasmid maps? Our lab standardly uses this software, and I would like to draw all of our new clones in this software.

I can create new entry clones (perform BP reactions) fine in VNTI, but when I try to perform LR reactions the software doesn't recognize the Destination vecotrs from the tol2 kit as destination vectors, although it does recognize that they have attb3 and attB4 sites. I am creating the VNTI files by pasting pDEST sequences from the Tol2 site. Any hints on how I get VNTI to recognize pDEST vectors?

Thanks,
Molly

jim lister said...

Hi Molly,

Instead of pasting the sequence into VNTI, try saving the annotated Genbank format sequence from the Wiki as text and then importing it. Alternatively, you might just check the file you already have to make sure the attB sites are annotated as "Misc. recombination" features, that might be the problem.

Good luck,
Jim

Chi-Bin Chien said...

Hi Molly!

Here's a comment from Jonathan Rosen back on 8/11/08:

----
Jonathan Rosen said...
I figured out the problem, which I will explain here in case anyone else has the same issue. Vector NTI wasn't recognizing the ccdB sequence in the vector, so I had to put it in manually by going to Feature Map --> add. After this, Vector NTI treated it as a destination vector.
----
I think the issue may be that there is a (nondeleterious) point mutation in the ccdB of all the destination vectors, which either came from Invitrogen in pDEST R4-R3 or was introduced at the very beginning of our multisite work. This single bp change prevents Vector NTI from recognizing the ccdB
--Chi-Bin

Chi-Bin Chien said...

Filipe--

You didn't say how big a construct you were trying to build. In general I would expect inserts up to 10 kb or so to work pretty well, and then would expect variable results above that. For very large constructs, we have used a construct p5E-FA that has 8-cutter (FseI, AscI) sites in a 5' clone, then clone in conventionally.

Chi-Bin Chien said...

Filipe--

You didn't say how big a construct you were trying to build. In general I would expect inserts up to 10 kb or so to work pretty well, and then would expect variable results above that. For very large constructs, we have used a construct p5E-FA that has 8-cutter (FseI, AscI) sites in a 5' clone, then clone in conventionally.

Chi-Bin Chien said...

Qing--

If your 5' clone is only enhancer, promoter, 5' UTR, and possibly intron (no start codon), the reading frame obviously won't matter; the starting frame will be set by the first start codon (presumably pME-EGFP or something similar).

We typically use proofreading Taqs (usually Phusion or the Takara long-amplification polymerase) that do not leave T tails.

Chi-Bin Chien said...

Clara-
There are some minor fluctuations in the att sequences, especially the longer ones like attR and attP. It is not clear whether these are errors in the Invitrogen documentation, or perhaps undocumented improvements from one version to the next of the Gateway kit. I just ignore single bp differences in the long att sites; everything still seems to work. However, if you are concerned, the p5E-MCS sequence is likely to come from direct sequencing and therefore to be correct, while the attR1 sequence comes from documentation and therefore is less certain to correspond to reality..

Molly Nyholm said...

Hi,
Thanks for the help getting VNTI to recognize pDest vectors (see 4/8/09)! FYI- Pasting the sequences works fine, but the attR3 and attR4 sites have to be annotated, and in the right orientation :).
-Molly

Anonymous said...
This comment has been removed by a blog administrator.
M'hamed Grati said...

Hi,
This Pr. M'hamed Grati,
I am starting learning about zebrafish transgenesis. I have a construct that I generated where I put the GFP under the control of a zebrafish cell type specific promoter/enhancer. I injected the linearized version of it into fish embryos, and obtained very sporadic cell type specific expression of the reporter, which is good news. Now, I want to border this construction with Tol2 5' and 3' sites. I wonder if you could provide me the primers (or full sequence) that I should use to amplify these fragments on zebrafish genomic DNA. Besides, I wonder if you could tell me where to obtain transposase mRNA for co-injection to zebrafish embryos. Thank you very much in advance for the help!
Please email me your feedback to m.grati@med.miami.edu since I rarely look at my Google account.