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.

Tuesday, April 6, 2010

Questions on the Tol2kit, 3/6/10-4/3/12

Ask general questions about the Tol2kit system by commenting on this post; we will do our best to answer in a timely manner. Reminder: please use your full name and institution, so that we know who is in the conversation. This posting covers questions from 3/6/10 onwards.

112 comments:

gerosenf said...

Hello,

I'm Gabriel from the Evans lab in Weill Cornell. I have a question regarding transgenic injection. I have been using miniprep DNA for injection into F0 fish to establish lines as I observed positives. I find that my efficiency in generating F1 founders is however very low and I am wondering if perhaps it is because I used miniprep DNA instead of midi or maxi quality. I've heard that midi or maxi is supposed to minimize protein carry over (potential RNases which would inhibity the transposase) leaving higher quality DNA. What do you guys normally use when you want to establish F0 generation fish.

Thanks for your time.

Chi-Bin Chien said...

Gabriel-
Midiprep/maxiprep DNA is probably somewhat cleaner than miniprep DNA, but we routinely use miniprep DNA for transgenesis, and it works well for us. (All of the experiments in the Tol2kit paper were done with miniprep DNA.) It is worth trying your injection +/- transposase RNA. You should see a big difference (more expression with transposase), which would confirm that your transposase RNA is good, and also that it's not being degraded during the process.

Also: you are injecting in the cell, and not in the yolk, at the 1-cell stage, right? It is probably worth injecting as early as possible: Kristen injects at early 1-cell stage, just as the cell is starting to rise up off the yolk.

Elizabeth 재은 said...

Hello,

I'm Elizabeth from the Zon lab at Children's Hospital Boston. I have been using your Gal4-VP16, UAS, and IRES-GFP constructs to make transgenics. I now have F0 GAl4 line, and F1 UAS-(middle entry)-IRES-GFP line. The good news is, the IRES-EGFP was expressed in the right region. However, when I picked green embryos at the gastrula stage and went back to them the next day, only small percentage of the IRES-EGFP animals had phenotype, whereas I also saw phenotype from the ones that I didn't pick the night before. Although it is still early, (since my Gal4 line was F0), it is still confusing that IRES-GFP did not correlate well with the phenotype. What has been your experience with the IRES-GFP transgenics?

Thanks for your time!

Chi-Bin Chien said...

Dear Elizabeth,

Thanks for the interesting question. I have to admit that we have not made many IRES-GFP stable transgenics (as opposed to just using IRES-GFP constructs in transient experiments). In fact, I can't think of any off the top of my head. This is partly because in the last couple of years we have tended to use viral 2A peptide constructs.

I suggest that you take some of the embryos with a phenotype, which you did not identify as GFP-positive the day before, and try both (1) antibody staining for GFP, and (2) genomic PCR for GFP.

These will test the possibilities that (1) some embryos did express GFP, but just at too dim a level for you to identify under the fluorescent dissecting scope, or (2) that the IRES-GFP cassette has somehow become silenced or damaged in some cases. You probably know Mary Goll and Marnie Halpern's results showing that the UAS often becomes methylated and silenced, which could easily cause embryo-to-embryo variations. Also, Shuo Lin has suggested to me a couple of times that the IRES may become silenced over generations. I think this has been seen in other systems, but don't have a reference.

Possibility 3: do you know that you are dealing with a single insertion of your UAS construct? With Tol2 transgenesis, we often find multiple insertions segregating in the offspring from a single F0 founder; different insertions can express at quite different levels due to position effect or perhaps silencing.

A final caveat: just because the F0 was injected with a Tol2 UAS construct and transposase RNA does not mean that all integrations are Tol2-mediated. Some fraction of transgenes will be due to simple plasmid integration, meaning that they will be rearranged concatamers in which the UAS-gene-IRES-GFP construct may no longer be intact.

Hope this helps. Please let me know what you find, I am very interested.

Melissa said...

Hello,

Is it possible to swap out and replace one element for another in a DEST vector easily? We received a DEST vector from a collaborator that was generated from 3 elements cloned into the pDESTTol2pA vector. Can I remove the promoter/enhancer (p5E) element and swap in another one to change the expression pattern of the fusion protein within that DEST vector? I do not have the pME and p3E vectors or this would be a piece of cake. It seems that this may be able to be done with BP clonase and ccdB tolerant bacteria (which can create single element DEST vectors) but am unsure.

If this strategy is not possible, could you give advice for removing the fusion protein, please? Would I need to design primers and amplify individual components or is their an easier way?

I'm in Bruce Appel's lab at the University of Colorado Denver. If there's an older post that addresses this comment, I apologize for having overlooked it.

Thanks for the help,

Melissa

Elizabeth 재은 said...

Thank you for your helpful comments, Dr.Chien. I will examine more fish, and also see what happens in the next generation. I will also examine how the IRES behaves compared to the transgene.

Thank you very much, and I will let you know how things turn out!

Elizabeth

Chi-Bin Chien said...

Melissa,
Yes, it is supposed to be possible to do a "reverse BP" reaction, combining your expression clone, pDONR P4-P1R, and BP clones, transforming into ccdB-resistant bugs, and selecting on chlor/amp, to get a new single-element destination vector. We have never tried ourselves, so I don't know how well it works. You could then do an LR reaction with the desired 5' clone to get a new expression clone.

Alternately, unless there are useful restriction sites, you would have to PCR out the middle and 3' elements to make new entry clones. (Can't you just get them from your collaborator?)

Good luck, please post if it works!

Jen said...

Hello,
I'm Jen Hocking from Reinhard Koester's lab. I've been using the Gateway kit to make multi-cassette UAS vectors, and have had moderate success. However, I have had some problems with almost all of the Donor vectors as well as the destination vectors...I find that they seem to just go "off" in the freezer and lose their ccd sensitivity (I make aliquots now, but even that doesn't always help). I would previously just retransform, prep several colonies and then test them by restriction digest and transformation into ccd-sensitive cells and I would obtain some useful preps. Lately I've been using the original pDEST R4-R3 vector because I only need transient expression and every prep that I make gives a lawn of colonies when transformed into XL1-blue cells. I noticed that there is now a pDEST R4-R3 II vector and I contacted invitrogen and they told me that the difference is a modified CmR-ccdB cassette for adjustment to the new E.coli ccdB survival strain (instead of the discontinued DB3.1 cells). Unfortunately, they refuse to sell me the new clone alone.
So my questions are: Does anyone have suggestions about how to avoid or deal with problems with destination vectors? I tested my prep of the pDestTol2pA vector by transformation into XL1-blue cells and it gave me a lot of colonies, but not a lawn and perhaps is still usable.
Does anyone have experience with the new pDEST R4-R3 II vector and does it solve these problems?
What does this imply for use of the pDestTol2pA vector with the available ccd-resistant cells?

Thanks very much for any advice.

Chi-Bin Chien said...

Hi Jen,

Don't know about the new version of pDest R4-R3. One point that might be relevant: our stocks of the original pDest R4-R3, as well as all of our destination vectors, whose ccdB-cam cassettes originate from the original pDest R4-R3, have a point mutation in the ccdB; my assumption has been that this mutation arose in Invitrogen's hands. However, all of the destination vectors and the donor vectors work much better in our hands than what you describe, with the exception of pDONR P4-P1R, which is problematical as noted on the wiki. Thus I don't believe that this point mutation is a problem.

It shouldn't really be possible for DNA to go bad in the freezer; instead, I would guess that that your DNA preps aren't completely pure, and instead represent a mixture of "right" and "wrong" (ccdB-mutated) clones. It should be possible to avoid this by proper microbiological practice. Is it right that you have generally just been running on minipreps of the donor and destination vectors, and propagating by retransforming every time? Perhaps you are giving evolution too much of an opportunity to act, and select crippled ccdB resistance genes.

Instead, what we do is to grow maxipreps (which last us a very long time, even with >10 people in the lab using them regularly). We propagate from glycerol stocks, streaking them out to make sure that we are really picking single colonies. We also make sure to grow all donor and destination vectors on both chlor and amp. In our hands, being careful about microbiological practice in this way has given us consistently good results.

I would suggest obtaining some good preps of all the donor and destination vectors to start with, testing colonies to select good ones, and making maxipreps and glycerol stocks. It might also be interesting to sequence the ccdB resistance gene in your existing clones; I predict that you will find point mutations (as we have occasionally found when we've had clones that went "bad").

Hope this helps.

lakshmi said...

Hello, Dr. Chien

I am trying to make a construct with 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

Chi-Bin Chien said...

Lakshmi:
In principle, sure, you could add a myc tag to the C-terminal end when making a middle clone by including it in the reverse primer (though the primer will be getting quite long). This gives up the combinatorial advantage of multisite Gateway though.

Why don't you just use 229. p3E MTpA? Make a middle clone without a stop codon, and using the standard Invitrogen convention for reading frames, and this will give you a 6x myc tag at the end of your protein. I'm not sure why you are worried about the attB3 sequence, as it will come *after* the 3' clone in the resulting expression clone. If you just want a single myc epitope, you could easily PCR from clone 229 to make a new 3' clone with 1x myc-pA.

PS: It would be preferable if everyone would identify themselves with their full name and lab affiliation.

Jen said...

Hi Chi-Bin,

Thank you for the advice. Looking back now (after 6 months off for mat leave), I realize that I only really had success with the pDestTol2pA2 vector, and so my pDestR4-R3 was maybe never very good. I did just do an LR rxn and the # of colonies following tranformation look promising. Of note, I do always use Chloramphenicol and try to pick single colonies. We typically make maxis of all our plasmids, and so I've done that with the Gateway vectors too, but I wasn't always happy with how they were working so I started to retransform them and do minis that I would then check by transformation into ccd-sensitive cells. You may be right then that I had a mixed population to begin with...whether from picking a mixed colony or getting a mutation while growing the maxi, I'm not sure. I do find that the minis tend to grow better and I do them now at 32C. I haven't really done mutliple rounds of tranformation to promote evolution, as you say. You're right that keeping a glcerol stock would be a good plan, and now I have some room to do that in our -80. I do think that all of these vectors have a tendency to mutate so one needs to be careful with them.
I talked more to Invitrogen and here is what they said about the new pDestR4-R3 vector:
"We discontinued our old ccdB resistent cells ccdB Survival T1R due to legal reasons and created a new strain ccdB Survival 2 T1R. The type of resistance of this strain is different to the old one which contained an overexpressed ccdA gene. Due to the differences in the resistence the ccdB cassette of some of the vectors had to be exchanged to allow our new strain to propagate this vector. Besides this change the vector is the same. "

lakshmi said...

Lakshmi Pillai(Morris Lab,University of Kentucky)

Hi,
What you said is right attB3 will be after the myc , I wrote B3 instead of B2. I will use the p3E-MTpA. I was worried about the B2 seq.(25 nucleotides).
I will follow you tip, and calculate the ORF.

Thanks for your time, i will let you know how this go..

Anonymous said...

Hello,

I'm Ethel, a PhD student at the University of Antwerp (Belgium). Please, I started making constructs and I already got the destination vectors. Even though, I can't start because I need the pDONRP4-P1R. Invitrogen sells this vector with the multisite Gateway cloning kit, which it's very expensive. Could you please send it to me?. I would really appreciate your help
Thank you

Chi-Bin Chien said...

Ethel--please email me directly for clone request (commenting anonymously does not provide any contact information). When you do, please let me know what version of the Tol2kit you have. v1.2 should have included all three donor vectors.

Adam said...

Hi, I'm Adam Navis from the Bagnat lab at Duke University. We have been using the Tol2Kit to generate constructs and wonder whether there is a p5E-UAS containing a fewer number of repeats, eg. 5x. We are concerned the many repeats could become silenced.

Thanks for your time.

Adam

Valerie Virta said...

Dear Dr. Chien,

My name is Valerie Virta and I'm writing from Merrill Hille's lab at the University of Washington. I am wondering if anyone has made plasmids that contain other fusion fluorescent proteins rather than mCherry or EGFP, such as YFP or CFP. I looked but I didn't see them on the list.

Many thanks,
-Valerie

Chi-Bin Chien said...

Hi Adam,

We don't have a p5E-5xUAS-E1b, though it would be a sensible thing to build (quite easy).

Does anyone out there have such a clone that they'd share?

Chi-Bin Chien said...

Valerie,

We haven't done YFP or CFP, but I'm sure others have.

Chi-Bin

Max said...

for Melissa and Others who tried "reverse" BP.

I tried reverse BP reaction. It is not very stable. but could give some hints:

1. You need to linearize vector before reverse BP (MluI).
2. You have to re-clone invitrogen destination vectors because most of them contain mixture of several clones. If you want to check it - digest with MluI+NruI and check picture. Or you will find it after sequencing of your target clone later.
3. use ccdB survival cells with two antibiotics. I used Chloramphenicol + Ampiciline (my clone has AmpR). Clones grow very slow. I usually grow them at 28C for 24h.

It is not working very well. I will be happy to know how to improve it. Please comment here!!!

Christopher said...

Hello,
I was wondering if someone created a gal4/vp16 fused with GFP? We are having issues with ires-gfp.

Thanks,

Chris Koceja
Ramshandran Lab at Medical College of Wisconsin

Max said...

Hi Christopher,

What kind of IRES do you use?
If you use pIRES from Clonetech it is not working at all.
Change it on something else gtx for example, or take it from any other working vectors.

Christopher said...

Thanks Max,

We are using the pME-Gal4VP16 followed by the p3E-IRES-EGFPpA element provided in the tol2kit but we do not see any gfp expression. We have verified gal4 is being produced. We want to use a gal4/vp16 fused with GFP instead the two previous elements as we have read the numerous issues involved with using IRES.

Chris Koceja

Max said...

Hi Christopher,

You better try T2A peptide: http://www.nature.com/nbt/journal/v22/n12/full/nbt1204-1590b.html

I made several vectors with it...

Jacob Best said...

Hello,
I am Jacob, currently working on a transgenics project in the Schulter lab at the University of British Columbia. I have the pT2HE vector with an HSP70 promoter driving GFP as a reporter (this is used for enhancer assays). I am interested in injecting embryos with a Tol2 construct containing both GFP as a reporter as well as another gene (bicistronic, not as a fusion protein). Does anyone have a construct that is more appropriate for what I am looking to do? Preferably something that has GFP with a promoter followed by a PolyA signal and then another strong promoter followed by cut sites for ligation of my gene followed by another PolyA signal. If not, then what would you recommend as the easiest way to build such a construct? Thank you very much for your time.

-Jacob

Chi-Bin Chien said...

Max--Please use your full name and affiliation; it would really be useful to know who is posting.

Chris--The IRES in the Tol2kit p3E clones is derived from a Clontech clone (pIRES2-GFP, if I remember right). It can work, as shown in our Tol2kit paper, but it is certainly weak and variable. Two suggestions:

1) Try anti-GFP antibody staining to amplify signal.
2) Make pME-Gal4VP16-no stop, and then use p3E-EGFP-pA to make the GFP fusion you were asking for.

Alternately, we have had luck with viral 2A interrupting peptides. See the Provost et al. paper from Steve Leach's lab a couple of years ago for use in zebrafish.

Chi-Bin Chien said...

Jacob--
I don't know what pT2HE is. Do you just want a GFP transgenesis marker? It doesn't sound like you're using the Tol2kit, but you could certainly use the pTol2CG2 destination vector to provide a cmlc2:gfp transgenesis marker, then put your enhancer in the 5' clone, and use pME-GFP or pME-mCherry as the middle clone, and p3E-pA.

Aaron Steiner said...

Dear Chi-Bin et al.,
This is Aaron Steiner in the Hudspeth lab at the Rockefeller University. I recently made a transgenic using the Tol2kit and am afraid that the beautiful expression pattern I am seeing might be due in part to position effects. I was wondering whether you or anyone else you know of has designed primers for TAIL PCR that are universal for Tol2kit inserts, allowing identification of insertion sites from any Tol2kit construct. I know that Parinov et al. (2004) did TAIL PCR on their Tol2 insertions, but it's unclear to me whether these primers will work with Tol2kit constructs as well.
Thanks so much,
Aaron

Chi-Bin Chien said...

Hi Aaron,

Position effect is certainly possible, especially if your expression construct is relatively small.

I can't remember how TAIL PCR works (I assume it's like LM-, splinkerette, or inverse), but it should be easy to check about the Parinov primers. Just take the gene-specific parts and check against the sequences for the destination vector you used.

As you can see by looking at the maps, pDestTol2pA2 and pDestTol2CG2 carry about 500 bp of the original Tol2 sequences at the 5' end (i.e., the downstream end relative to R4-R3) and 200 bp at the 3' end (i.e., the upstream end).

So, there is a good chance that the Parinov primers will work. When you figure it out, can you please post the answer here?

kn56375 said...
This comment has been removed by a blog administrator.
kn56375 said...

Hello,

I am Kyle Nelson and I am working in a start-up lab under Ted Zerucha at Appalachian State University and we have little funding. We are attempting to produce transgenic lines for enhancer analysis using the older Tol2 construct pT2AL200R150G and are having significant trouble cloning putative cis-elements in a XhoI upstream site and BglII downstream site. We have tried everything but can't seen to get the ligation reactions to work. We have also had trouble transcribing transposase mRNA directly out of the plasmid but were successful off of a PCR product. I was wondering what bacterial lines you would recommend for growing the Tol2 plasmid as well as the transposase construct (pCS-TP)? I was also wondering if you are aware of any methylation issues that are associated with these plasmids? Any information would help.

Thank you so much for your time.

Chi-Bin Chien said...

Hi Kyle,

As far as I know, there aren't any issues with cloning into these Tol2 vectors; what you are trying should work. The standard controls (e.g. ligating vector only as well as vector plus insert) apply. Make sure you aren't UV'ing any more than necessary when you gel-purify. Et cetera. Neither XhoI nor BglII are dam- or dcm-methylation sensitive. Pretty much any standard bacteria should work.

As for making RNA, it's odd that the PCR product works but not the plasmid. I assume that you are linearizing the plasmid with a single-cutter that cuts after the polyA sequence.

Sorry not to be more help. Good luck.

Chi-Bin

Fang said...

Dear Dr. Chien
My name is Fang Lin at University of Iowa. We are trying to make transgenic lines using your vector with the cmcl2:GFP marker. We made many lines successfully. Thank you very much for sharing the reagents with us.

However, this time the promoter is only 140bp (was published by other
group). We made the 5'-entry promoter clone using pDONR P4-P1R and the sequence is correct, but we failed to get any 3 way LR clone. We have done test LR reactions using different 5'entry, middle entry and 3'entry clones and found out the problem is our 5'entry clone. I suspect the problem is
that the 5-entry is too small to have recombination.
I am wondering if you or any reader have any experience on that. I would like to know if it is possible to get such 5'enty work. Should I try traditional cloning instead?
Thank you very much in advance.

Max said...

Hi Fang,

My shortest insert was 82 bp and it work well. As a way you can try another compitent cells with higher transformation level. I prefer cells from Zymo Research.

Could you like to give references on such small promoter?
Is it constitutive promoter?

Chi-Bin Chien said...

Hi Fang,

Please remember to leave your full name and affiliation, I think it helps the discussion.

I agree, the problem is not size per se. We have done quite a few LRs with a 5' entry clone that has an 84 bp insert, with no problem. There may be some sequence-specific problem with your clone (e.g., if there was internal sequence that looked like an att site).

As a positive control, you can use p5E-Fse-Asc (included in the Tol2kit). This is very short, but should work. If you continue to have problems, you can do the LR with p5E-Fse-Asc in place of your clone, and then use the 8-cutter (FseI, AscI) sites to conventionally clone your 140 bp fragment into the expression clone.

Max said...

Hi Fang,

I forgot to mention. I had problem with one my vector before. I resolved problem by titration "problematic" vector.

I took all compounds and add "problematic" vector in different concentrations: 1/4, 1/2, 1, 2, 4 from recommended. In my case 1/2 gave best result. After first titration I used this vector in 1/2 for all other reactions.

Max

Fang said...

Hi Dr. Chien and Max

Thank you very much for the tips. We are going to try p5E-Fse-Asc as well as Max's suggestions on better competent cells and the dilution trick. Will update our results.
The promoter we are trying to clone is the cxcr4b promoter, which was published by Christine Dambly-Chaudièrea's group in PNAS 2010, 107:6358. Your comments on promoter itself is interesting. We will look at that. Max: what do you mean "constitutive promoter"?

By the way, I was Lila Solica-Krezel' postdoc at Vanderbilt (I got the full set of gateway clones from her lab). Now I have my own lab at University of Iowa.

Thanks again for helping us out.

Fang

Anonymous said...

Hello,

I’m Elizabeth Gibbs from Eva Feldman’s lab at the University of Michigan. We’re having some problems with transgenic fish generated using a vector with the cmlc2:egfp marker.

We have identified a number of lines with bright fluorescent hearts, but none seem to be expressing our gene of interest. I have screened positive F1 transgenics from 12 different chimerics, and while I can always detect the presence of my gene by genomic PCR, I cannot detect expression by RT-PCR or western blot.

This forum had a conversation related to this issue several years ago (late 2008), and I was hoping to find out if this is a common problem with the cmlc construct. Are we likely to identify positive carriers if we keep screening? (We are using the beta-actin promoter, not the hsp70 promoter described in the original publication.)

Thank you for your help, and thanks for a great resource!

Chi-Bin Chien said...

Elizabeth--
After 12 founders I would probably give up. We have seen and heard of problems with the cmlc2 promoter interfering with the main expression construct; however, we haven't seen this for the bactin2 promoter, which is strong. Is it possible that expression of your gene of interest may be deleterious? bactin2 drives quasi-ubiquitously, including in eggs before fertilization; therefore, if expressing your protein compromises viability, there will be strong selection for silencing of the bactin2:(your gene) cassette.

Perhaps trying the hsp70l promoter would make more sense--little basal expression, and strong ubiquitous (quasi-ubiquitous?) expression upon heatshock.

alex said...

Hi
I have a technical question about
pDestTol2 plasmids. We have a number of these clones that are between
14 and 16 kb in size (total size). And for many of these we have a really tough
time getting a decent yield. All we get is 10-50 ng per ul from our
minipreps. Midipreps often fail and give nothing. Have you ever run
into a similar problem?
Thanks,
Alex Nechiporuk
Oregon Health & Science Univ

Chi-Bin Chien said...

Alex--

So, you mean that your final expression clones are 14-16 kb total size? We have actually gone up to something like 22 kb. I assume that you are just want them for injections, and therefore need ~10 ug (that is, midipreps were just an effort to get more DNA).

As far as I know, we have not had the yield problems that you describe. Are they dependent on particular genomic sequences that maybe the bacteria don't tolerate?

The problems that we have instead had are that certain genomic fragments like to recombine when we do the LR reactions and transform. What we have taken to doing is to use CopyCutter electrocompetent bacteria from Epicentre. These are bacteria that propagate colE1-ori plasmids at low copy number, unless you induce with arabinose (?). We transform in these bugs, grow plates at room temperature, pick colonies, and do normal minipreps (we don't bother with the high copy induction). We get enough DNA from these minipreps to do diagnostic digests; at the lower temperature and low copy there seems to be less selective pressure, so that we get correct, unrearranged clones. We then take DNA from a good mini and retransform into regular bugs to do a normal miniprep and get a reasonable amount of DNA for injecting.

So my suggestions are:
1) Maybe try the chemically-competent or electrocompetent CopyCutter bugs? Perhaps the induction will help?
2) You could try growing cultures at lower temperature. However, if I remember right the colE1 ori is temperature-sensitive, so that at lower temperature the copy number is lower (not what you want).
3) Have you tried Terrific Broth or something similar?

Finally, is this actually an elution problem? For columns like the Qiagen gel-extraction column, we find that fragments over 10 kb are hard to elute. Qiagen recommends warming the elution buffer to 60 degrees or so; we sometimes even warm up the whole column. You could try a phenol-chloroform miniprep just to test how much DNA is really there.

Hope one of these ideas helps. Let me know--I'm curious.

Max said...

Hi Alex,

Do you have problem with Destination vectors? If so, Dest vectors contain ccdB gene - suicidal gene. You cannot get high copies amount. Now you can use for them only ccdB survival cells from invitrogen.

We have such problem. We grow cells at 25-28C for ~24h. We are using LB media. SOC gave larger chance of recombination and loosing vector.

For DNA extraction we are using Zymo miniprep-Classic. It is several times cheaper than Qiagen. And they give us more stable results. Be careful, their columns has capacity around 15 mkg/column. For dest clones we cultivate cells in 15 ml of media and using 2 Zymo columns and elute in 60 ul of hot (65C) Extraction buffer. In result we are usually get 120 ul of DNA with concentration 100-200 ng/ul.

arul said...

Hello all,
I am trying to create a transgenic line containing BAC construct with promoter sequence. Ideally, I would like to use Tol2 repeats for the transgenesis. However, the BAC sequence is about 70Kb. I was wondering if Tol2 would work for such large sequences? If not, would I-Sce method be a good alternative?
Thank you for your time
Best wishes

Chi-Bin Chien said...

Arul--
Koichi Kawakami's lab has published a paper on the use of Tol2 ends for BAC transgenesis:
Transposon-mediated BAC transgenesis in zebrafish and mice.
Suster ML, Sumiyama K, Kawakami K.
BMC Genomics. 2009 Oct 16;10:477.

Shirley said...
This comment has been removed by the author.
Shirley said...

Hi,
I recombined the 5'entry clone, middle entry clone and p3E-EGFP into pDEST-Tol2pA. The final construct has the fragments from 5'entry clone and middle entry clone. The embryos injected with the final construct fluoresced. When I sequenced my construct, I discovered Kanamycin resistance gene in my final construct. The Kan gene seems to come from p3E-EGFP. How is this possible? Moreover, this construct can't be cut with XhoI and BglII. Any idea of what is happening?

Chi-Bin Chien said...

Dear Shirley,

It sounds like you made the 5' and middle clones. Do you know that they are correct? Usually we do a restriction digest, and also sequence with M13F and M13R if necessary to rule out PCR errors.

I assume that you meant pDestTol2pA2, not pA.

There may be some unexpected products that can result from LR reactions, but normally they will be eliminated by appropriate antibiotic selection. When you do the LR reaction and transform bacteria, you should be plating them on an amp plate, no? In this case, the resulting clone should be amp resistant, not kan resistant.

Shirley said...

Dear Dr Chien,
I sequenced my final construct. The sequences of the fragments I cloned into the 5' and middle entry clone are correct. I used ampicillin agar plate and this colony grew on it. However, when I used a primer to read into the EGFP fragment brought in by p3E-EGFP, I found the Kanamycin gene downstream of my attB2 site. I assumed that EGFP is also in the construct since the embryos fluoresced. I am wondering with the p3E-EGFP has recombined with the reverse strand and hence Kanamycin gets into my final construct. Will the kanamycin resistant gene have any effect on my GFP expression? Thanks a lot.

Chi-Bin Chien said...

Shirley--

Sequencing is not particularly useful as a diagnostic except for entry clones that have been built using PCR. Restriction digests are much more useful for looking at overall structure of the clone. I would test entry clones as you build them; you may reuse them in the future for different LR reactions.

Presumably your expression clone with the kanR also has ampR; very likely it also lacks Tol2 transposon ends, and who knows what the overall structure is. Given this uncertainty, it is unsuitable for use in experiments.

Did you really only pick one clone from the LR reaction plate?? You need to pick multiple colonies (4 or 6 at least), design a suitable diagnostic digest, and pick a clone that gives the right bands.

virginie said...

Hello,

I'm Virginie (Lecaudey lab, Freiburg, Germany). I have generated quite many transgenic using the tol2 kit in the last 3 years but recently we had 3 examples of founders (with completely different transgenes) which show extremely weak green fluorescence in the heart, so that it is barely detectable. Genes are expressed under hsp70 or UAS and these founders also seem to express our transgenes at an equivalently low level after induction (heat-shock or when crossed to GAL4 driver line). I have never seen that before and have no clue what is going on. I would be very interested to know if somebody already had the same problem.
Thanks a lot for your time.
Best,

Virginie

Chi-Bin Chien said...

Virginie--
I don't think we've seen that particular problem. Normally I think of hsp70l as being a strong promoter (perhaps the strongest that we know of), so this is surprising. Is the problem sporadic (occasional cases interspersed with normally-behaving transgenics), or does it seem instead that it is happening every time? I'm just wondering if you could have a bad prep of the destination vector (with a mutation or rearrangement).

virginie said...

Hi Chi-bin,

thanks a lot for your reply. We have that now with 2 hsp70 and 2 UAS lines that we have done recently so you're right that we should maybe prep again our pDEST. But we are always using the same pDEST from the start, can something really happen when it's kept at -20°C?
Best,

Virginie

virginie said...

Hi Chi-bin,

again concerning these very weak bleeding heart associated to weak transgene expression under hsp70 control. We have now sequenced and checked by digestion our pDEST and everything looks fine. Since mainly the hsp70 transgenes seems to be affected by this weak expression anyway, I have the impression it is not coming from the pDEST (we are also doing a lot with UAS and it looks fine in this case). We have sequenced our p5E-hsp70 as well of course and it is fine. And anyway, it seems that there is a clear correlation between very weak bleeding heart (which is independent of hsp70) and weak expression of the gene controlled by hsp70. Do you think it could be a question of number of insertions? Is it known if there are multiple insertions of the transgene with the tol2 system? Could it be that for any reason (different transposase?), we have now less insertions for each transgene? We have indeed 2 different transposase, one coming from the tol2 kit, one coming from the Kawakami lab if I'm not mistaken and they are slightly different (one has a Kozak sequence, and the other does not seem to have a polyA). I would be very grateful if you could lgive us your opinion about that.

Chi-Bin Chien said...

Virginie-
If you read Koichi's papers, it is clear that one should expect multiple independent insertions with Tol2 transgenesis, at least when using the modern versions of the vectors and transposase. Koichi's original transposase clone did not have a Kozak sequence or a conventional polyA signal, which is why we rebuilt the transposase clone for the Tol2kit, putting it into pCS2. Insertion number is presumably correlated with transposase activity, so if you have a bad batch of transposase RNA, or unknowingly reduce the injected volume, you could indeed get fewer insertions.

As for why you are getting weak cmlc2:gfp expression, as well as weak expression of your genes from the hsp70l promoter, I am still mystified. Many people have reported instances of interference between the cmlc2 element and the main expression cassette. The idea is that the cmlc2 element not only promotes expression in the heart, but also suppresses expression elsewhere. In some cases it appears to act at long range and silence your main construct. It is surprising to me that the hsp70l promoter should be silenced in this way, since it is normally so strong, but it is certainly a possibility.

Some questions:
1) Do your recent hsp constructs, which express weakly, have anything in common, e.g. long coding sequences? We have certainly noticed that it is harder to express coding sequences longer than about 2 or 3 kb.

2) Did you have older hsp constructs that worked well?

Joachim Bentrop said...

Hi,
Using the alpha-actin promoter to drive we find only punctate expression of our genes of interest in the ventral part of the myotomes (36 hpf), not a uniform expression. Is that commonly observed?
thanks for your help
Joachim
(Bastmeyer lab)

Chi-Bin Chien said...

Joachim-
You do mean alpha-actin, not beta-actin, right? We have very little experience with this promoter. However, punctate/variegated/mosaic staining is of course very common in transient injection experiments.

Does alpha-actin:GFP give you the same kind of punctate staining? This may just be how this promoter behaves in transients; perhaps it is especially susceptible to position effect.

sfsoccergroup said...
This comment has been removed by a blog administrator.
Meej Kim said...

Hello,

I'm Meej from the Ahituv lab at UCSF. I am about to perform the LR Clonase II reaction with the P5E, PME and P3E plasmids into pDestTol2pA.
My question is regarding the actual LR Clonase II that is used. The protocols on the Tol2Kit site recommend the LR Clonase II Plus (Cat#: 12538-120) enzyme. Is it possible to use the LR Clonase II enzyme(Cat:11791-020)? If not, what is the difference between the two reaction mixes?

Thank you.

Chi-Bin Chien said...

Meej-
You need to use the enzyme mix specified in the protocol, or things won't work. The composition of the mixes is proprietary (otherwise they couldn't be so expensive!).

Zachary Gaber said...

Hello,
My name is Zachary Gaber, a grad student at the Novitch Lab at UCLA. We've just starting getting into the the Tol2kit and have been having some strange problems with culturing bacteria transformed with what I thought were successful LR clonase II plus clones.

I transformed the LR clonase II plus reaction into Invitrogen competent Top10 cells, grew on LB+Amp plates, picked colonies, and tested for the proper inserts through diagnostic digest AND a PCR strategy with one primer inside the insert and another primer on the vector. Both digest and PCR confirm that I got the clones. I had kept a mL of the bacteria from the original minipreps used these very same cultures to innoculate an LB+Amp maxiprep culture. Same LB, same antibiotic, same cells the grew perfectly in miniprep cultures are growing pathetically slowly in maxiprep cultures. After 14 hours in the shaker at 37 degrees, the cultures just look slightly cloudy! Is there any special treatment that pDestTol2 clones require. I know about ccdB and Cm, but these successful clones shouldn't have either! Any help would be appreciated. Thanks.

Chi-Bin Chien said...

Zachary--you are right that your trouble with growing maxipreps is perplexing. As you say, your final clones should be amp-resistant, ccdB-, and should no longer have chlor resistance either. I suspect that this is not a Tol2 or Gateway-specific problem, but just a general problem that one sometimes sees with growing up certain plasmids: overnight cultures for minipreps grow well, bigger cultures for maxipreps don't. I'm sure that there are reasons for this that lay outside the range of my microbiology knowledge.

You should perhaps check that your shaker is set properly and that you're following recommendations for the culture size (typically something like 50 or 100 ml). Otherwise, I would just try growing for longer. It is only a few doubling times from slightly to very cloudy. If this doesn't work, retransform from your miniprep DNA, and try starting again from a colony.

greenfish said...

Hi Dr Chien and all,

I am Zhiqiang, from Liz Patton's lab at the Edinburgh Cancer Research Centre. Wellcome to the 7th European Zebrafish MeetingEdinburgh, Scotland 5th - 9th July 2011

I have a question about the reverse BP reaction. I am trying to replace a cDNA with chlor/ccdB cassette on a gateway construct. The vector is believed to be derived from pTolDest with R1/R2 sites. I did a BP reaction with this plasmid, pDoNR221 and BP clonase, transformed into ccdB survival cells and grew cells on Amp/chlor plates. I could only see tens of colonies growing from 50ul/300ul transformation. The colonies were very small and grew slow. When I sequenced the colonies with sp6 and T7 primers, only see the chlor resistance gene from T7, not ccdB gene from Sp6. Sp6 didn't give signals, the primer binding site (or/and attR1 site) seemed to be lost. I used the resulted 'destination vector' for single-way LR, did not obtain any colony.

I just read Max's comment on July 12,2010 saying that the vector should be linearized with MluI. Are there any other improvements? Can anybody share his/her experiences on reverse BP reaction?

Max said...

Hi greenfish.

I am doing reverse BP on regular basis now. It is work perfectly.

the protocol is:
1. digest pDONR vector with MluI+NruI (or MluI + EcoRV)
2. Cut from a gel fragment 3000 kb
3. If possible leniarize second vector.
4. setup BP overnight
5. transformation to ccdB survival,
6. plate with Chloramphenicol only
7. grow at 30-37C at least 24h.
8. large colonies -wrong, small is correct.
9. for miniprep you will need to grow around 24 hours. Yeld of vector is small. I usually grow in 15 ml LB with Chloramphenicole. Yeld is 10-20 mkg


I usually pick 2 small colonies and sequence ends for one of them.

good luck

Chi-Bin Chien said...

Max--
Thanks for leaving your detailed protocol, very useful. It would still be nice to know your full name and affiliation.

Interesting that you need to grow at lowered temperature. The colE1 ori is temperature-sensitive, so growing at 30 degrees should give you low-copy rather than high-copy growth. We often use this trick for large plasmids (e.g., an LR reaction giving a 22kb product), but it is not normally necessary for the pDONR or pDest vectors.

greenfish/Zhiqiang--
As Max points out, it is important to linearize the pDONR plasmid to reduce background, since it has the same resistance as your desired plasmid.

If your plasmid is indeed derived from the Lawson lab's pTolDest, it is not surprising that your SP6 doesn't work--this vector does not have an SP6 site. Instead, use a reverse primer directed against the SV40 polyA signal sequence.

I'm confused about what you're doing though. Shouldn't your pTolDest-derived plasmid, plus pDONR221 in a reverse BP reaction, give you pTolDest back? Wouldn't it be easier just to get the original pTol2Dest plasmid?

Max said...

Hi Chien,

I prefer do not give my affiliation yet.
The 30C cultivation could be overkill for your vectors. Vectors which I am using stable only in the Stbl3 or HB101 cells (contains large repeats). In others they could grow only at 28-30C without recombinations.

Max

greenfish said...

Many thanks to Max and Dr chien!

Sorry, the expression vector is not derived from pTolDest, and it contains a promoter, I want to replace the existing cDNA with other cDNAs. The attB sites are flanked with T7 and Sp6 sequences, which can be used for sequencing of cDNA.

It seems that only attB2 was recombinated with attP2 in all 4 colonies sequenced.

Normally BP reaction is very efficient, I did not expect to have such difficulty.

It is a great and very helpful website. Thanks for your time and effort.

Anonymous said...

Hello Tol2kit community,

Does anyone have access to the following destination vectors?

- pDEST™14
- pDEST™15
- pDEST™17
- pDEST™24

These are listed under the "E. coli Expression System with Gateway® Technology" (catalog no's. 11801-016, 11802-014, 11803-012, 12216-016).

We have a collaborator here in Halifax would would like to use Gateway to grow up a high quantity of zebrafish protein in E. coli to help prepare a new antibody. Unfortunately, these vectors are prohibitively expensive...

Does anyone have access to these vectors? If yes, would you consider sharing a small aliquot to aid our work?

Alternatively, does anyone have experience using a *different* pDEST vector to grow up protein in E. coli?

Any advice would be greatly appreciated!

Many thanks,
-Michael Forrester
Dalhousie University (Halifax, Canada)

Lakshmi said...

Hello Dr. Chien,
My name is Lakshmi from Uni. of Kentucky, Dept of Biology. I identified a correct clone for hsp70I-gene-IRESEGFP, although I see green hearts upon heat shock @37, 38 (multiple trials) I cannot see expression of my gene of interest. I raised the founders and checked for green hearts in the babies, but no luck there either.
I was thinking may be this is happening since I did not do BP cloning; i put my gene of interest int he pME-MCS vector and performed the 3 way LR cloning step.
I was wondering If I can by-pass the LR cloning and put the gene of interest directly into the destination vector between the Tol2 sites. If not the do you think I should do BP cloning and them LR cloning.Please do help me with this issue.

Thank you
Lakshmi
Dept.Of Biology
UKY

Chi-Bin Chien said...

Michael--
Sorry, don't have experience with other extremely expensive Invitrogen destination vectors. Presumably you could take non-Gateway E. Coli vectors and Gatewayize them by putting the key features into a p5E- or p3E- clone, and then just use any destination vector, perhaps pDestR4R3, to build your E. coli expression clone.

Chi-Bin Chien said...

Hi Lakshmi,
I'm a bit confused: please clarify what your construct is. hsp70l-gene-IRES-EGFP should give ubiquitous expression, not green hearts, upon heatshock. Perhaps you were using pDestTol2CG2 to include a cmlc2:egfp marker? This would give green hearts independent of heatshock.

As for using pME-MCS to build middle clones, you do have to be careful (as indicated on that clone's page). It was not designed very well, and there are late stop codons in two reading frames. Basically, there is only one useable reading frame. With this caveat, it should work fine; but BP reactions are even simpler!

Lakshmi said...

Hello Dr. Chien,

Sorry for the confusion, you are right about the destination vector. I did not see any any expression post heat shock. May the stop codons are the reason.

The other question was to insert the gene of interest along with a promoter directly into the destination vector, bypassing the LR reaction. Do you think it will work.

Best Regards
Lakshmi
UKY, Biology

Chi-Bin Chien said...

Hi Lakshmi,
Is your reading frame such that you have an in-frame downstream stop codon? If so, this will certainly prevent expression of the IRES-EGFP.

As for doing conventional cloning instead: of course you can do this, but there are few useful restriction sites since the destination vector wasn't optimized for this.

Finally, it is worth using antibody staining against EGFP to increase sensitivity; you should also use confocal or compound microscopy, not just a fluorescent stereomicroscope.

Chi-Bin

arul said...

Dear Dr. Chien
I have cloned a muscle specific promoter region into a p5'Entry vector recombined with pME GAL4FF and p3'E polyA into pDESTCG2 destination vector. I am wondering if CMLC2 GFP has a leaky expression in the somites. I notice a few fibers that glow green in the somites.
Thank you for your time
Best wishes
Arul

Lakshmi said...

Hi Dr. Chien,
Thank you for the ideas, will try the antibody and no I don't have a in-frame stop codon, but for sure I don't see heat-shock induction in the transients or the F1 offsprings.

Thank you.
Lakshmi

Chi-Bin Chien said...

Arul--

So you have a construct:

msp:Gal4FF-pA; [cmlc2:egfp-pA]

where the square brackets indicate opposite orientation.

The possibilities are:
1) Unregulated GFP expression in muscle in transients (quite common)
2) msp promoter may crosstalk to express the EGFP
3) cmlc2 promoter may be showing leaky expression of EGFP

There are quite a few control experiments you could do, but it's not really worth debugging the transient experiments too much, since they often show nonspecific expression. I wouldn't worry unless the problems show up in stable transgenics.

Max said...

Arul,

Suppression of expression is happened often if you use several promoters in close distance.
the solution is put 2 elements between them.
I used before following constract:
Promoter1-gene-pA-Tactb-cHS4-Promoter2-gene-pA

where Tactb- is transcription terminator from actin B. cHS4 is gene insulator HS4 from chicken.

In you case probably will be better to put Tactb-cHS4-[Tanother one].
I do not have time to find references on this construct now. If will be able not early before next Monday.

Good luck!

Lakshmi said...

Hello Chien,

I am trying to place a promoter directly in the pDest CG2 vector and wanted to know the positions at which I can successfully do that.

Thank you
Lakshmi
Dept. Of Biology
Uni. Kentucky

Chi-Bin Chien said...

Hi Lakshmi,
It is unclear exactly what you are trying to do. The complete sequence for pDestTol2CG2 is available on the wiki, which will let you find any useful restriction sites.

I do think you are off in the wrong direction. LR cloning is much simpler and should be able to do anything you want. You need to
(1) detect the IRES-EGFP as sensitively as possible, i.e. after anti-GFP staining on a good compound scope or confocal.
(2) carry out a control experiment, like hsp70l:mCherry-IRES-EGFP-pA. This should give you blazing mCherry expression after heatshock, and you can then test out your assay for the IRES-EGFP.

Lakshmi said...

Dear Dr Chien,

I will follow your advice and do a control LR cloning to look for hsp70I induction. And also try the whole animal antibody staining for GFP. Thank you very much for being so helpful. I will keep you posted as to how things work out.

Sincerely,
Lakshmi

Deepak said...

Hello,

Would anybody here be willing to provide a new P4-P1R DONR vector? We haven't made any new 5' clones in a generation of graduate students and now it appears those set of cell stocks / plasmid tubes have been misplaced.

I'd be much indebted. =)

Deepak
Biological Engineering, MIT

Carlos said...

Hello,

Does anyone know what size is the largest insert that can be cloned into a destination vector? Would 20kb be reasonable to attempt?

Thank you

Carlos said...

Hello,

Does anyone know what size is the largest insert that can be cloned into a destination vector? Would 20kb be reasonable to attempt?

Thank you

arul said...

Hi everyone.
I had cloned a potential promoter element of the gene of my interest in the 5' donor vector to yield a p5E-promotor clone. I cloned this p5E-promoter clone with pME-GAL4 and p3E-PolyA into the pDESTCG2 vector by multisite LR reaction. The final destination clone was injected into the eggs and babies were raised. The founders obtained from these babies (screened by virtue of their CMLC2::GFP expression) were grown and crossed with uas::kaede lines. The embryos were screened for specific expression. I expected the expression to be in differentiating myotome based on teh insitu expression pattern. I obtained sufficient quantity of embryos with bright green hearts. The following were my observations:
From pair#1, 25% of green heart embryos (GHE) showed expression in the myotome, a few notohord cells and neurons of CNS.
From Pair#2, 25% of embryos showed expression in notochord, few neurons of the CNS and few isolated muscles.
From Pair#3, 30% of embryos show expression in spinal chord, rhombomeres, few cells of the eye, Olfactory epithelium and fin bud but no expression in the muscles.
I have the following questions with respect to my observations:
1. Why are only a few of the GHE showing other tissue specific expression patterns.
2. How come each of the transgenic founder (essentially carrying the same transgenic contruct) show different expression pattern compared to the native insitu expression pattern of the gene?
All suggestions to help me understand these results will be gratefully appreciated.
Thank you for your time and best wishes

Chi-Bin Chien said...

Carlos--
We have successfully made stable transgenics with inserts (between the Tol2 sites) of about 19-20 kb. The limit at that point is the cloning capacity of the vector (~25 kb for pUC-based plasmids).

Chi-Bin Chien said...

Arul--
There are two basic issues.
First, you are getting variegation/silencing of the UAS:Kaede transgene. It is now well-known that this happens quite strongly with the UAS sequence, probably because of the two CpG dinucleotides at either end, which are targets for methylation. So a cell that has Gal4VP16 may not show Kaede.

Second, it seems to me that your "promoter" is not very well characterized? Ideally one hopes to clone out a genomic fragment and have it contain all the key enhancer and promoter elements for the gene, but this may not happen. I would certainly use a yourpromoter:gfp-pA construct as a first test of how good it is. You are clearly getting a lot of position effect, i.e. the genomic context strongly affects the expression from your promoter. If it is truly a "promoter", you would get no expression from promoter:gfp and in fact you are doing an enhancer trap experiment!

Green heart just means that the embryo carries DNA from your construct; it does not guarantee how the construct will express in that genomic context.

Mengqi said...

Hi,

I am Mengqi from Ralph Martin's lab in West Australia. I have a question about the efficiency of IRES. I have generated a construct with my target gene (with stop codon but no polyA) followed by IRESGFP, however, the expression of GFP is almost undetectable. The promoter I used is a neuron-specific HuC promoter, which is a quite strong promoter when used in my other constructs. I am wondering if you can give some suggestions on it. Thank you.

Max said...

Hi Mengqi,

What kind of IRES did you use? The Clonetech has 2 different IRES. The IRES from pIRES did not work at all in our hands yet IRES2 worked pretty well.
I recommend you find a vector which carry our IRES2-GFP, check it is work or not, and after this use for cloning.

Mengqi said...

Thanks for replying, Max. I used the IRESGFP from p3E-IRESGFP-pA (389)vector. Since I have already got my interested gene under the HuC promoter in a mini tol2 element based vector, I didn't really bother to use the gateway system, but just grabed the IRES-GFP-pA from vector389 and ligated it after the stop codon of my gene. What would you think might cause the problem? Thank you very much.

arul said...

Dear Prof Chien and Max,
THank you for your insight into my observations. I will try to verify the promoter based on your suggestions.
Arul

Chi-Bin Chien said...

Mengqi,

As described in the Tol2kit paper, the IRES we used is IRES2 from Clontech (which Max declares superior). I have also now added this information to the individual clone pages.

As described in the Tol2kit paper, the IRES drives expression more weakly than the first cistron. How much more weakly seems to depend on a couple of factors, including how long the first cistron is. How long was your gene of interest? In our hands, once the first cistron is longer than about 2 kb, the expression of IRES-GFP seems to drop quickly.

A final factor to consider is how you detect the IRES-EGFP. We always antibody stain for EGFP (to amplify the signal), and take care to image on a good compound scope or confocal scope.

A useful control might be to build HuC:nlsmCherry-IRES-EGFP. This should give about the brightest IRES-EGFP that you can with the HuC promoter, and give you something to compare your experimental construct to.

JAKE BLOG said...

Hi All,
I'm Jake from the Appel lab and Univ. of Colorado - Denver.
I've noticed a trend away from Gal4VP16 and toward Gal4FF in the literature. Many have used VP16 successfully. If FF is indeed preferable though, would anyone out there be willing to provide our lab with a plasmid containing Gal4FF such as pME-Gal4FF?

Thanks much

Jake Hines

jacob.hines@ucdenver.edu

Thomas Frank said...

Hello,

my name is Thomas Frank from the Friedrich Lab in Basel. We are currently using the Tol2Kit to create UAS responder lines, so far all being based on the pDestTol2CG2 vector. After reading some of the comments on this forum I became a bit worried that the cmlc2:EGFP cassette could potentially interfere with the expression of our genes of interest. Specifically, I would like to ask whether the notion "(...)we have only tested the cmlc2
promoter (...) with UAS-driven reporters" in the Kwan et al., 2007 paper refers to a direct injection of UAS constructs into Gal4-expressing embryos (where the UAS/E1b sequence would act as a strong promoter) or into wildtype embryos (where the sequence should act as a 'weak' or rather silent promoter). We are currently raising UAS responder (F0) founder fish (on a WT background), and fear that - in addition to the known problem of silencing of UAS-controlled sequences - the clmc2 promoter might have an additional, negative effect on UAS controlled gene expression after we cross F1 responder fish to Gal4 driver lines. Does anybody have experience with this or with a similar setting?

Many thanks in advance for your feedback!

Best regards,
Thomas

Thomas Frank said...

Hello,

I do have another question, on which I would greatly appreciate any feedback. It has been mentioned on this forum that people have (succesfully) used 2A constructs for bicistronic expression - rather than IRESs. Has anybody used a middle entry clone with a 2A sequence (e.g. Provost et al., 2007) (and obviously no STOP codon), followed by a 3' entry clone such as YFP? And if so, what would be your general impression of the 'danger' that (a) the extra C-terminal amino acids encoded by the 2A sequence and/or (b) the extra N-terminal amino acids of the 2A sequence and the recombined att site adversely affect the function of the 'middle' and/or '3'' protein, respectively? Obviously this might depend strongly on the actual proteins, but any comment about general trends or succesful examples would be very helpful.

Again, many thanks in advance.

With best regards,
Thomas

Dru said...

I have used the Tol2 system for generating transgenics for a year now, but I'd like to clone a new promoter into a 5' entry vector. The promoter is currently in PCR8 and I have tried a few times to amplify the 5 kb promoter and ligate it into the 5' MCS entry vector. I have no problems amplifying the promoter (which has restriction sites on the primers), restriction digesting the insert and vector, but the ligation just won't work. I see that you used a BP reaction to clone in the bactin promoter into a 5' entry vector. What exactly is that BP reaction and can I get an aliquot of the 5' entry vector? Thanks so much,
Dru

Dru said...

Nevermind, I see the Protocols are on the Tol2 wiki site. Now I need to find out how to get a sample of pDONR P4-P1R and BP clonase II.

greenfish said...

Hi Max,

I am still strugling with reverse BP reaction. I followed your protocol but have never successed. My questions are: 1) I used BP clonase II, should I use BP clonase instead? 2)what is the proper ratio of two vectors? how much DNA do you use in a 10 ul reaction? 3)My destination vector will be Amp/Chlor resistance, should I plate cells onto Amp/chlor plates after transformation? 4) I tried to grow cells at 32C and 37C, no colony grew. Should I try even lower temperature?

Thanks a lot again.

Zhiqiang

Max said...

Hi Zhiqiang,

1) I used BP clonase II, should I use BP clonase instead?

BP clonase II

2)what is the proper ratio of two vectors?
1 vector: 2 insert
Overnight reaction!

3) how much DNA do you use in a 10 ul reaction?
same as in Invitrogen Protocol (I don't remember exactly)

3)My destination vector will be Amp/Chlor resistance, should I plate cells onto Amp/chlor plates after transformation?

Chlor only.

4) I tried to grow cells at 32C and 37C, no colony grew.

I used 25-28C and grow 24-48hours.
You need to pick only small colonies (they are transparent and almost invisible). Large are with mutation in ccdB and will not work for LR later.

Please contact me directly next time: http://www.linkedin.com/in/maxmikheev

Anonymous said...

Hi, I'm Jake from the Appel lab @ Univ. of Colorado Denver. I'm wondering if anyone has tried to make destination vectors with alternative markers to cmlc2:EGFP, such as cmlc2:cherry or gammacrystalline? This was mentioned to be in progress by Kwan et al. in the 2007 Developmental Dynamics paper but I can't find any further information. A second independent marker would be extremely helpful to identify double positive embryos from Gal4-UAS crosses. I would be very grateful to hear from anyone who has generated this, or tried and found what doesn't work.

Thank you,
Jake
jacob.hines@ucdenver.edu

Anonymous said...

Hello,

I'm Adam from the Schilling lab at Univ. of California - Irvine. We have had a lot of success with the gateway system, but I have run into a problematic BP reaction. I am attempting to put a large amplicon (~10kb) with attb1 and attb2 sites into the pDONR221 vector and we cannot get positive clones. Other BP reactions have worked great for us but several tries with this one have yielded no success so far. I believe people have said they can clone in 10kb amplicons with not a lot of problems, so are there any recommendations we can follow to help increase the chance of success? Thanks for any help you can give!

Robert G. Hesse said...

Hi,

I'm Rob from the Pomerantz lab at UCSF. I successfully created multiple Tol2kit transgenesis vectors as well as several founder fish for each one. While I'm waiting for the the fish to reach sexual maturity, I want to characterize the transgenic protein. I have tried transient transfections in ZF4 cells with my transgensis vectors (lipofectamine, fugene, and nucleofection), but I do not get very good efficiencies. Do you think that I will get better efficiencies if I perform co-transfections with the Tol2 transposase plasmid (Tol2kit#396)? If so, what ratio of transgenesis construct:Tol2 plasmid do you recommend? I am also in the process of creating stable lines of ZF4 cells with my constructs.

Thanks and best regards,

Rob

Anonymous said...

Hello,
I'm Rob from the Neuhauss Lab in Zürich. I'm using the Tol2Kit to generate transgenic zebrafish lines which express GFP under the control of promoters of our genes of interest. So far I have had success.

Now I am trying to blindly clone the upstream region of a gene up to 10 kb in search of the promoter of the gene of interest. This causes some problems: After optimizing my PCR (Temperaturegradient etc.) I always get an amplification of a really big fragment (basically stays in the slot, so big it is). I repeated the setup with genomic as well as with BAC DNA as a template but still I get the same result...
Of course I now think about designing new primers (again), but first I wanted to know, since the attprimers show secondary structures, if it possible that my amplified fragment of interests somehow clusters together through the attsites resulting in big fragment size or so?

Furthermore: What Polymerase do you use for big fragments (I have been using the Phusion Polymerase as suggested in the paper, should work, no?)? Any special treatment of your template DNA?

Thanks a lot for your time and your advice!

Max said...

iProof from BioRad is the best.

chunlin said...

Hello, I am Chunlin from TLL at singapore. I am starting using the tol2kit in the lab, however, the A2 and CG2 vector I've got seem not correc when I degested with pvu II, they both give a small band around 400bp and a large band. Is there someone nearby, and could share some ul with me.

Temasek Life Sciences Laboratory
1 Research Link, National University of Singapore
Singapore 117604

Kristen Kwan said...

Hi everyone,
Kristen Kwan here - I apologize for our long absence from the blog. As many of you may know, Chi-Bin passed away in December after a long battle with cancer. I will be working to respond to each of your questions now - thank you so much for your patience!
Best,
Kristen

Zachary said...

Hello,
My name is Zachary Gaber, a graduate student in the Novitch Lab at UCLA. We're trying to clone a neuron specific enhancer into the pDONR-P4-P1R and it's been surprisingly hard. No colonies yet. I can amplify a band and have done the usual controls - I think the problem is just that I don't have enough DNA in the tube. I'm probably at about half the recommended number of moles and that's if you can believe my nanodrop at such a low concentration. I'm a bit scared of introducing mutations by just scaling up the number of cycles (we're already at over twice the number of cycles I usually use for BP cloning) and was wondering if I could increase the time if the BP clonase reaction instead. Overnight is mentioned in the manual, but could even longer help more? What is the longest that you you've let a BP clonase reaction go and were there any issues with going too long? Thanks for any help you can give me.

Kristen Kwan said...

Hi Zachary,
We haven't let BP reactions go longer than overnight. (I imagine the clonase will lose activity over time anyway.)

One possibility - I don't know what volume you're using for your reactions, but I've performed BP reactions in as little as 5 ul. Another option is to simply set up two identical PCR reactions, and load both over the same gel extraction column to double the amount of DNA.

One note is that the pDONR-P4-P1R vector is the one particularly problematic donor vector; it can undergo self-recombination. Are you sure that you have a good prep of donor vector? A protocol to test the vector is available on Nathan Lawson's lab website (http://lawsonlab.umassmed.edu/gateway.html).

Good luck!
Kristen

Zachary said...

Thanks for your quick answer. I am reasonably sure we have at least a decent prep of pDONR-P4-P1R as we have used successfully before, but that may be an issue causing reduced efficiency. Maybe just doing 2xPCR reactions and reacting over night is the answer. Thanks!

chunlin said...

Hello, this is Chunlin at TLL singapore. I've put pME-mCherryCAAX into pDest for RNA transcription and microinjection, however, I can not detect cherry expression. I send this construct for sequencing and find there is a C-G mutation cause His to Asp AA change at position 80.
Does it happen with your constructs too?? Is this point mutation really matter for expression??
Thank you

Kristen Kwan said...

Dear Chunlin,
You have the old version of pME-mCherryCAAX (Chien lab clone #232). That mutation does indeed destroy mCherry fluorescence, unfortunately. It was fixed and has been superseded by Chien lab clone #550. This is all documented on our wiki.
Does your lab (or anyone else at TLL) have the Tol2kit update v. 1.2? The new mCherry-CAAX clone was distributed in that update. If not, please email me your address (kristen.kwan@neuro.utah.edu), and we will send you the filter.
Best,
Kristen

洪春姿 said...

Hello, i am hygia:
if our middle entry clone is small, it would be more quickly to got the our final density construts to succeed? and as small insert how smallest number is suggest? Thank you

Anonymous said...

Hello, I am Tuanjie Chang,
I used pDestTol2CG2 (#395) to make several destination constructs, and want to confirm the inserted sequences. I used M13R primer, but later found there are two M13R primer sites on this vector, which messes up the sequencing results.
Is there anyone sequenced the insertion in above vector? please share the sequencing primer. Thank you.

Bharath Gopalan said...

This is really informative. Thanks and keep up the good work
stereomicroscope Singapore