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, June 8, 2009

Questions on the Tol2kit, 6/8/09-3/5/10

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 6/8/09-3/5/10.

54 comments:

Jim Dowling said...

I am interested in generating a transgenic from a large cDNA (roughly 18000 bp). Is there any size restriction with the Tol2 system? Also, how difficult is it to introduce Tol2 sites into an existing plasmid

Anonymous said...

Greetings Dr. Chien:

Have you had any luck using Evrogen's tagRFP in zebrafish? We were thinking of trying it as a general "red" tag in zebrafish because of the limited brightness of mCherry; however, we've come across one or two blog postings elsewhere on the web where the person posting claimed that tagRFP does not work in zebrafish.

Your insights would be most appreciated.

Regards,

--Leon Parker

Chi-Bin Chien said...

Jim--

We have successfully made stable transgenics with inserts of about 19-20 kb. Presumably once you add a promoter and polyadenylation signal you will be above 20 kb, but I think it should work fine. The limit at that point is the cloning capacity of your vector (~25 kb for pUC-based plasmids).

It's not difficult, though tedious, to introduce Tol2 sites into a plasmid by conventional cloning. You would just have to PCR up each end with appropriate restriction sites, then ligate it in.

Chi-Bin Chien said...

Hi Leon,

We have indeed made several transgenics with TagRFP, and it is blazingly bright--brighter than EGFP on our fluorescent dissecting scope. These are cytoplasmic TagRFP; we haven't done much yet with fusions.

In my experience, statements of the form "X works in flies/worms/mice but not in zebrafish" almost always turn out to be false.

Matan Golan said...

Hi all,
I am having problems with the pDONRP4-P1R. I spread cells (from a glycerol stock from another lab) on kan+chloram LB plates. I picked six colonies and purified plasmids. My problem is that when I transform them into ccdB intolerant cells (DH10B or DH5alpha) I still get some colonies growing. For example, if I transform ~200ng of plasmid I get 40-60 colonies per clone in most clones and in one clone I get a full "carpet" – but in none of them do I get zero clones!!!
When I did the recombination test suggested in Lawson's lab page I got better results (probably due to much less plasmid used for transformation) with 2 clones giving zero colonies but I still don't understand why I get colonies using untreated plasmid.
Anyone had similar results? Any suggestions?
Thanks in advance,
Matan.

Chi-Bin Chien said...

Matan,

ccdB is not a perfect selection (not as strong for instance as antibiotic selection), so it is not surprising that you get a few colonies. It is also possible that a few molecules are undergoing self-recombination, excising the ccdB, and thus giving colonies.

In any case, the most relevant control is the Lawson lab recombination test--this is the closest to the actual BP reaction that you will be doing.

Successful BP reactions with pDONR P4-P1R will give you hundreds of colonies, of which only a few are empty vector. Picking 4 colonies, digesting, and sequencing almost always gives us the desired clone.

In summary, it sounds like you have some good preps of donor vector, and you should go ahead and try the real BP reaction.

wnorton said...

Hi Dr. Chien,

I am having trouble amplifying the putative promoter regions for a couple of genes of interest. The fragments I am interested in are not so larger - between 6kb and 3.5Kb

I would like to ask, which method do you use for preparation of high quality genomic DNA, and can you recommend any particular polymerase for the PCR?

Many thanks

Will Norton

Jing said...

We are new to zebrafish and are in a process of making a transgenic fish. I have successfully amplified 10kb upstream of ATG (which I assume contains the promoter) and would like to use this to drive mcherry expression.
I plan to clone this 10kb fragment in p5E-MCS and then use pME-mcherry and a p3E-SV40 polyA. Is this the correct direction to go.
Kindly advice me on whether the extra sequences introduced by the MCS would harm the expression of mcherry.

Quint said...

Hi Dr. Chien,

I have attempted two BP reactions to clone a 5 kb insert into the pDONRP4-P1R vector overnight and transformed the products the following day on kan plates. The next day, I came in to find anywhere from 6-75 colonies on each of the four plates and I screened close to 50 of those colonies. I did not observe anything that resembled a successful clone.

In the 10 ul BP reaction step, I used 35 fmol of the insert and followed the exact concentrations of everything else in Invitrogen's instructions for the reaction. Does anyone have any advice or experience with something the size of or bigger than my 5 kb insert, and, if so, is there anything else I could be doing to get successful clones?

Many thanks for your help in this matter.

Quint Reid

Chi-Bin Chien said...

Hi Will,

We don't do anything particularly fancy when preparing the genomic DNA. Proteinase K digestion followed by either a phenol/chloroform cleanup or else DNAzol should work.

For amplifying long fragments, we've been using TaKaRa LA polymerase.

Chi-Bin Chien said...

Jing--

Your strategy is a good one. 10 kb upstream of the ATG is indeed likely to contain the promoter, unless there is a noncoding 5' exon and an intron >10 kb before the first coding exon.

Note however that this 10 kb will not necessarily contain the tissue-specific regulatory regions that you are probably looking for. Such enhancers can be close to the transcriptional start, but can also be 50 or 100 kb away, either up or downstream.

Chi-Bin Chien said...

Quint,

My guess is that you have a bad prep of pDONR P4-P1R. This donor vector is rather unstable (prone to self-recombination), and needs to be propagated very carefully (as stated on the wiki).

If you have not yet successfully completed a BP reaction to make a 5' clone, it would be best to find a neighbor who has (and therefore has good pDONR P4-P1R), then carefully regrow the donor vector from their stocks.

Michael Mattocks said...

Hello Dr. Chien,
I'm using the Lawson lab protocol to prepare a good pDONR p4-p1R prep. When I transform ccdB resistant bugs with my p4-p1R stock, I get two fairly distinct colony sizes, a few large and many small. The multisite gateway manual notes that this is symptomatic of mutations in the ccdB gene in BP reactions - is this the case with the Lawson lab selections as well? Should I only chose large or small clones? Thanks in advance!

Anonymous said...

Hello Dr Chien,
I want to to use a PCR product of 9kb in the gateway system. Then use the multifragment gateway to generate a final construct of 13kb. Do you think there will a problem working with such large entry clones?

tx in advance,
best regards
Pedro Campinho

Chi-Bin Chien said...

Michael--

I'm not sure whether we have paid attention to small vs. large colonies for P4-P1R. If you're not sure, I would just pick some of each.

This is the hardest clone in the whole kit to grow up, so it is worth taking care at this stage.

Chi-Bin Chien said...

Pedro-

We have successfully done multisite LR reactions with entry clones whose inserts are longer than 9 kb (in our case, an enhancer-promoter element). It is probably not quite as efficient as for smaller pieces, but is certainly doable.

The problems that we have sometimes seen with these longer clones are likely due to the fact that they are genomic sequence containing multiple repeats, which are thus prone to recombination.

When we've had trouble, we've used a simple trick: transform the LR reaction, spread the bacteria on a plate, and then grow up at 30 degrees (or room temperature if 30 degrees isn't convenient). The colonies will grow more slowly, but at these lower temperatures the pUC ori drives significantly lower copy number, so that there is less selective pressure for rearranged/shortened plasmids. We pick colonies and grow liquid cultures as normal (at 37 degrees), though if you were worried you could also grow the cultures at 30 degrees.

For very hard cases, we have used electrocompetent CopyCutter bugs from Epicentre. These give very efficient transformation, and then only replicate the plasmids at low copy number. You don't get as much DNA from your minipreps, but once you have the right clone, you can retransform into regular (DH5alpha) bacteria to grow up larger amounts of DNA.

Using these methods we have made Tol2 constructs with inserts as large as 19-20 kb.

Anonymous said...

Dear Dr.Chien: I have cloned 10Kb fragment in TOPO XL T/A cloning vector. Can I make primers with attL4 and attR1 and generate a entry clone. The att sites seem to be quite long for a primer. Is there any simpler way to add allL4/R1 sites to generate entry clone.
Thank you - Jing

Chi-Bin Chien said...

Jing-

You should really read the Gateway manual. Or the wiki. Or the Tol2kit paper.

Indeed attR and attL sites are too long to add using PCR primers. Instead, add the appropriate attB sites, then do a BP recombination with pDONR P4-P1R, to get the entry clone.

Molly Nyholm said...

I am looking for 5' entry clones containing ubiquitous promoters other than B-actin.If anyone has others that they are willing to share, I would be greatful. Otherwise, I will be making them, and will share when they are complete!
Thanks,
Molly Nyholm
Grinblat lab

Chi-Bin Chien said...

Hi Molly,

There is the h2afx promoter from the Tol2kit, which John Parant cloned. I haven't used it myself; looking at the expression patterns on ZFIN, I suspect that it is expressed in proliferative zones rather than being truly ubiquitous.

It would be great to have something as good as the ROSA locus in mouse (though I believe even that isn't perfect). I don't know of any other ubiquitous promoters, sorry; I wouldn't be surprised if someone else has made one though.

Is there a particular reason that you're unhappy with the bactin2 promoter? I have certainly heard anecdotal reports that it is not truly ubiquitous, but we have not observed this ourselves.

Molly Nyholm said...

Chi-Bin--
We might give h2afx a try, but were hesitant because of the "quasi-ubiquitous" label.

We are trying some in vivo enzymatic protein tagging, would like to have a couple of different ubiquitous promoters, ideally of different strengths. We were thinking of making an Ef1a 5'entry clone(stronger?) and comparing it to Bactin2 (weaker?). Someone also suggested that we try the tubulin promoter. Any input on which of these might be most useful is welcome.
Thanks,
Molly

Chi-Bin Chien said...

Hi Molly,

The Xenopus EF1alpha promoter might potentially work, but I don't know if it's stronger or weaker. I have never liked it that much because it turns off after about 18 hpf. I don't know if anyone has cloned the zebrafish EF1alpha promoter.

You could also do things like crippling the Kozak consensus sequence in the bactin2 promoter--this is pretty likely to give you weaker expression, with exactly the same pattern.

To be honest, though, the simplest thing is just to generate a few insertions with your bactin2 construct. You are likely to get enough position effect that different insertions will give significantly different levels of expression. You'll have to clean up for a couple of generations to isolate single insertions, but you'd likely have to do this anyway.

Or, even if all bactin2 insertions were exactly the same, you could just vary copy number. For instance, insert1/+ vs. insert1/+;insert2/+ vs. insert1/insert1 vs. insert1/insert1;insert2/insert2.

jim lister said...

Hi Molly,

I agree with Chi-Bin that your best bet is probably generating multiple lines with the same promoter and selecting those with different expression levels due to position effects.

And I just wanted to add in regard to the ubiquity of various promoters, definitely take a look at Burket et al (2008) Transgenic Research 17:265 and Thummel et al (2006) The Scientific World Journal 6:65.

-jim

mki3 said...

Hi All-
We're just beginning to use the Tol2 system - we'd like to make a hsp-Cx43-IRESEGFP clone. For preparation of the middle element using pDONR221, do you recommend including the 5'UTR for the Cx43 gene or can we begin with the AUG?
Thanks!
Kathy Iovine

Chi-Bin Chien said...

Hi Kathy,

I usually assume that if the 5' UTR is in the range of 50-200 bp with no secondary structure, translation will be fine. In practice, we tend to strip off the native 5' UTR and just start with the AUG, though I would definitely add a Kozak consensus sequence beforehand: CCACCaug. Between the attB1 site and the fragment of the hsp70l 5' UTR that is in the hsp70l 5' clone, this should give you a decent-length 5' UTR. The hsp70l promoter is very strong and always works well for us in such a configuration.

Natasha said...

Dear Dr Chien,

I am trying to do an LR reaction with the following inserts: an 8.8kb 5', 3.3kb M, and 200bp 3'. I am using the pDESTTol2pA2 (394 clone) destination vector. I have tried the reaction 3 times and each time I get colonies carrying ~5-6kb and 3kb plasmids that seem to correspond (in size) to my original destination and entry clones. I'm surprised to be consistently getting individual clones carrying multiple plasmids, none of which correspond to my expected plasmid (~16kb). Could you comment on what you think might be happening?

Thanks,
Natasha Novikov

Chi-Bin Chien said...

Natasha--

I'm not quite sure what "getting individual clones carrying multiple plasmids" means--are some colonies 6 kb and some 3 kb?

First let me ask--have you successfully done LRs before? You are selecting on amp plates, right?

Raquel C. said...

Hi Dr. Chien,
I was wondering if the tol2 constructs that are generated to inject in fish have been used in pronuclear injections to generate transient mouse transgenics. I was wondering because initially I was testing mouse sequences in zebrafish for enhancer function, but am now worried about the degree of conservation of the entire locus I am looking at since a published enhancer for our gene of interest didn't show any reporter expression in fish. I was thinking of using the same constructs I had generated to test enhancers in fish and injecting them in mice. I know that Koichi Kawakami published a paper in October of this year of generating mouse transient transgenics with BAC's using Tol2, but I haven't found anything about smaller constructs. Any info is greatly appreciated!
Really appreciate your help,
Raquel Castellanos

jim lister said...

Hi Raquel,

I wonder if there is a cell line you could transfect to test your constructs -- at least that could tell you if your cloning went right and your reporter can be expressed, and it would be way simpler than making transient Tg mice. A negative result wouldn't be very helpful but a positive result would save you a lot of work.

-Jim

Chi-Bin Chien said...

Raquel--

I haven't heard of anyone using the Tol2kit to make constructs for mouse transgenesis, but I strongly suspect that it will work. After all, it's just a set of cloning reagents, not a zebrafish-specific technique. Presumably if you provide transposase activity at the same time, you will increase integration rates and also have the chance of making clean single-copy insertions.

Good luck!
Chi-Bin

Anonymous said...

Hi Dr. Chien,

We are new to zebrafish and are in a process of making a transgenic fish.We are makeing oncogene fused EGFP or mCherry by multisite Gateway technology.In the case, Promoter-EGFP-oncogene or promoter-oncogene-EGFP.While one, I can doing. Beceuse,in my case.pTol-Promoter-EGFP-mCherry or pTol-Promoter-mCherry-polyA.There can working very well(30-40%).But,pTol-promoter-oncogene-EGFP.It does not work.I was maked 4 plasmids,only one plasmid can work.The Success Probability is very lower(5%).So,I was maked new plasmid by IRES. pTol-Promoter-oncogene-IRES-EGFP.There do not working.I was injeted 75pg or 25pg DNA with 25pg Tol2 RNA.I can founed Fluorescent.Anyone had similar results? Any suggestions?

Thanks in advance,

JW.

JW said...

Hi Dr. Chien,

We are new to zebrafish and are in a process of making a transgenic fish.We are makeing oncogene fused EGFP or mCherry by multisite Gateway technology.In the case, Promoter-EGFP-oncogene or promoter-oncogene-EGFP. That one,I can doing. Beceuse,in my case.pTol-Promoter-EGFP-mCherry or pTol-Promoter-mCherry-polyA.There can working very well(30-40%).But,pTol-promoter-oncogene-EGFP.It does not work.I was maked 4 plasmids,only one plasmid can work.The Success Probability is very lower(5%).So,I was maked new plasmid by IRES. pTol-Promoter-oncogene-IRES-EGFP.There do not working.I was injeted 75pg or 25pg DNA with 25pg Tol2 RNA.I can not founed Fluorescent.Anyone had similar results? Any suggestions?

Thanks in advance,

JW.

Ali said...

Hi,

I received an HA and myc tag from a lab along with the pDONR221 and pCSDest2 vectors and was wondering if you might know the antibiotic resistance of the tags and whether they contain ccdB in their vectors?

Thank you.

Jeff Yoder said...

Hi Chi-Bin,

Do you know of anyone who has built a 3' entry clone with an IRES seqeunce and dsRed (as apposed to GFP)?

Thanks
Jeff

Chi-Bin Chien said...

JW-

I am having trouble understanding your question. If I understood properly, a potential problem is that IRES-EGFP will express quite dimly. Perhaps try screening again allowing for this possibility? A higher zoom on your fluorescent dissecting scope may make things look brighter.

Chi-Bin Chien said...

Ali-

You haven't provided much information about the HA and myc "tags" (plasmids?). We have not used HA; there is one myc clone in the Tol2kit (p3E-myc-pA), whose sequence is available on the wiki site.

Chi-Bin

Diane Jones said...

Dear Dr. Chien,

We are using a construct with the hsp70 promoter. Can you advise us as to the concentrations of DNA and transposase RNA we should inject? Also, can you give us more information as to how to heat shock the embryos? We have placed the embryos at 37 for 1 hour, but haven't seen any bright green from our gfp protein. We have looked right after heat shock, and after 24 hours. Maybe we are not injecting enough DNA and RNA?? If you could give us some ways to trouble shoot, I would appreciate it.
Thanks so much,
Diane Jones

Chi-Bin Chien said...

Diane-
Injecting 1 nl of 25 pg/nl DNA and 25 pg/nl RNA is a good place to start. You were unclear about your constructs. I suggest making hsp70l:egfp-pA as a positive control construct. This should give very strong, ubiquitous expression in the embryo/early larva. Heatshocking for 1 hour at 37 degrees should work well. Ideally you want the temperature to change fast. Pipetting the embryos into a tube of prewarmed medium is a good way to do this. You should then see maximal GFP expression at 4-6 hours post heatshock.

Anonymous said...

Hello Dr. Chien,

I am working with a genomic piece of DNA that I would like to add directly into the Tol2 expression construct without an p3E SV40 late polyA signal or a p5E donor. Essentially I'd like to use the pME-MCS with my genomic dna and add it to the pDestTol2pA2. Is it possible to do this?

-Brian

Chi-Bin Chien said...

Brian-
pDestTol2pA2 is meant for use with 3-insert LR reactions. Nathan Lawson's lab did make a Tol2 destination vector designed for use with 2-insert LR reactions. In either case, you'll need to use empty or "stuffer" entry clones in the unused positions.

For pDestTol2pA2, you could make a 3' clone that contained a short or neutral sequence, then use this together with a middle clone containing your genomic fragment, and p5E-MCS in the 5' position.

Brian said...

Hello Dr. Chien,

I had an alternative thought about the tol2kit for adding a single vector. Would it be possible to add the pDestTol2pA by a BR reaction if I added the attL4 and the attL3 to the ends of my genomic fragment?

-Brian

Brian said...

Sorry Dr. Chien,

I meant a BP reaction.

-Brian

Anonymous said...

Re: Brian's question, I think I've got a P3P4 donor vector in my freezer somewhere. This should let you put a single fragment into the R3R4 destination vector. Email me at adam dot rodaway at kcl dot ac dot uk, and I'll try to dig it out.
cheers,
Adam

Chi-Bin Chien said...

Brian--
In principle, adding L3 and L4 sites to your clone would work. However, these sequences are very long, probably too long to add using oligos. So if Adam Rodaway really has a P3-P4 donor vector, doing a BP vector with it would be the way to go.

But still, using "stuffers" in the 5' and 3' positions is not that hard.

オテモヤン said...
This comment has been removed by a blog administrator.
greenfish said...

Hello,

Does anyone have the experience in cloning the flanking sequence of a transgene by inverse PCR? Would it be possible that you share the protocol with me?

Zhiqiang Zeng

Chi-Bin Chien said...

Zhiqiang--

We have done some cloning of flanking sequences, but I don't have a good protocol written up. Try some of Koichi Kawakami's papers--there should be a protocol there.

Diane Jones said...

Dr. Chien,

We have created a control hsp-EGFP construct, injected embryos, heat shocked them the following day via your suggestion of pipeting them into warmed (37C) water for an hour, and then viewed them 4-5 hours later. We have not seen any green. We believe our transposase RNA is fine. We have checked it on a gel. We are using 25pg DNA and 25-30pg RNA. We have sequenced the promoter and that is correct.

We are hoping that you can help us trouble shoot some more. Should we try increasing the amount of DNA? Any help you can provide will be appreciated.
Thanks,
Diane Jones

Anonymous said...

I am wondering if anyone has generated a 3'clone containing the viral 2A sequence followed by mKate, CFP, or mCherry?
Thanks,
Kathleen

Chi-Bin Chien said...

Diane-

I can't think of a reason why your hsp:egfp controls are not working. I assume that you have tested the final expression construct by digest and/or sequencing? Even if the transposase RNA is not working, straight plasmid injection of hsp:egfp should give you lots of green after heatshock. Do you know that your microscope filters work well for visualizing GFP? Is there another zebrafish lab nearby that you could compare notes with?

Julian.- said...

Hi Dr. Chien:

I was wondering if someone has made a p3E-UAS-mCherry (or EGFP)-PolyA plasmid.
The idea is to use it with pME-Gal4-VP16 and p5E-MCS with my promoter.
I was going to make it myself and discovered that there is no restriction site (or room) between the attR2 and mCherry in the p3E mCherry polyA vector.
Do you know if anybody has tried adding some 15 or 20 bases (as a spacer with restriction sites) using the "All around" PCR strategy (as in site directed mutagenesis?
I thought that this could be a faster and easier strategy than cloning all 3 fragments and religating them. Instead I would add unique sites to insert the UAS fragment from the p5E-UAS.
Does this sound like a feasible strategy to you? Any suggestions?

Thanks a lot...

Julian Sosnik.-

Chi-Bin Chien said...

Hi Julian,

Responses to your questions:

--We have not made (or thought of making) p3E-UAS-mCherry-pA.

--Sorry that p3E-mCherry-pA is not easy to clone into; it was designed to make a clean C-terminal fusion of mCherry and constructed by PCR and a BP reaction.

--Your suggestion of adding an upstream restriction site by site-directed mutagenesis, then cloning in a UAS fragment, should work. Note that you will also want to add a proper Kozak consensus to the mCherry (it does not have one).

--Alternately, you could use fusion PCR to amplify (10xUAS) and (Kozak-mCherry-pA), adding attB sites to the ends, then use a BP reaction to make the desired 3' clone.

--Note also that you would need to add a polyA signal to the middle clone: pME-Gal4VP16-pA. (We may actually have this in the freezer somewhere...)

lakshmi said...

Greetings Dr. Chien

I am trying to make a construct a 3'myc fusion tag. I was concerned that the att B3 sequence in the expression vector might shift the myc out of frame.
I was contemplating adding the myc tag seq. to the reverse primer of the gene of interest, before the stop signal in the middle entry clone .

Thank you very much..

Best Regards
Lakshmi

A. Victor said...

Hii
i want to get the multigateway tol2 kit vector. How can get it ?