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. We encourage you to post with your full name (not just your first name) so that we have some idea who is asking.
30 October 2007: There has been a fair amount of spam recently, so I have turned on word verification for comments. You will have to do a "word recognition" task before you can post a comment. Please email me directly if you have any problems with this. --Chi-Bin
25 comments:
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
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
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.
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.
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.
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.
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
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.
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
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.
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.
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.
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!
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
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.
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.
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
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.
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
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.
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
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.
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
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
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.
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