tag:blogger.com,1999:blog-7158647951423191205.post9215350112304182072..comments2023-09-29T05:46:05.022-06:00Comments on Tol2kit: Questions on the Tol2kit, 4/23/07-6/14/07Chi-Bin Chienhttp://www.blogger.com/profile/04543802635501556885noreply@blogger.comBlogger37125tag:blogger.com,1999:blog-7158647951423191205.post-7725211992568663242014-04-09T11:18:03.461-06:002014-04-09T11:18:03.461-06:00Hi, I'm designing primers to amplify a promote...Hi, I'm designing primers to amplify a promoter that I will use to make a p5E. I was wondering if it makes any difference to the expression of the pME if the p5E includes ATG in the sequence? Or should I design the reverse primer before the ATG?Linanoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-22238147974743827132011-03-01T10:31:54.649-07:002011-03-01T10:31:54.649-07:00Will LR Clonase II work for multi-fragment gateway...Will LR Clonase II work for multi-fragment gateway or do i need the LR Clonase II Plus ?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-45264206712407156802008-09-09T09:42:00.000-06:002008-09-09T09:42:00.000-06:00hi,i'm going to use your pME-mCherry clone and was...hi,<BR/><BR/>i'm going to use your pME-mCherry clone and was wondering if there is a difference between nlsmCherry and H2AmCherry and if one of both gives a better signal than the other? (we want to use it for cell tracking)<BR/>Thank you!sarahhttps://www.blogger.com/profile/06853473449999730668noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-74938650355980471242008-09-07T14:21:00.000-06:002008-09-07T14:21:00.000-06:00Dear Chi-bin Chien,First, I also wanted to thanks ...Dear Chi-bin Chien,<BR/>First, I also wanted to thanks all the people involved in developing and sharing this system and constructs.It is great!. Thank you; Chi-bin Chien, Kristen Kwan,Ben Mangum and Esther Fujimoto... also for making a really useful web and blog. Also to the people that did the apE programm. <BR/><BR/>I have some problem with the IRES-caaxEGFP. I made a construct carring the pt2(1.5hspGal4VP16IREScaaxEGFPpA) using the the 5',ME and 3'entry clones that are in the tol2 kit and the pDestTol2pA2(394) also from the kit. I checked the sequence and seem to be all right however when I injected this construct to make and small enhancer trap, from the mosaic EGFP positive embryos, quite a lot have strong EGFP in the yolk from the end of gastrulation on wards. The yolk starts getting black (cell death?) and by 48-72hpf lot of them dye. Did you find any similar thing using the caxx-EGFP?.I have try different concentrations and seem to happen the same. The strange thing is that the injected embryos that do not express EGFP in the yolk survive well. Before I injected embryos with the same concentration of pT2600bphspGal4VP16pA (without any Ires memebrane EGFP)and I did not have this problem. To my knowledge no one has reported leaking or basal expression of 1.5hsp in the yolk. Is that right? Did you try any minimal promotor (not the c-fos) that works well(not basal expression)in zf?.<BR/>Thanks again for all your help.<BR/><BR/>AmayraAmayrahttps://www.blogger.com/profile/11876099766796068196noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-90306899963933574342007-08-31T06:30:00.000-06:002007-08-31T06:30:00.000-06:00Hi Tom,I have seen maternal deposition of GFP with...Hi Tom,<BR/><BR/>I have seen maternal deposition of GFP with mothers that are stably transgenic for several promoters, including H2A, beta-actin, and EF1alpha. Never tried alpha-actin, but I'm not surprised at this result.<BR/><BR/>We often propagate these lines using fathers since it simplifies the screening.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-25770108607872914582007-08-23T01:40:00.000-06:002007-08-23T01:40:00.000-06:00When I use a beta or an alpha actin promoter with ...When I use a beta or an alpha actin promoter with mCherry, in the offspring from my G0's I get a huge amount of red accumulating in the early blastomeres, presumably from maternal transcript.<BR/><BR/>The majority of these eggs lose all expression over the next few days and only a small proportion are germ-line.<BR/><BR/>I've never seen this with GFP. My ideas are:<BR/>i) maybe this also happens with GFP, but you can't see it because it's not as bright<BR/>ii) maybe the mCherry message is somehow more prone to maternal deposition<BR/>iii) maybe all of the red eggs are germ-line, but extensive silencing occurs around the MBT in the G1.<BR/><BR/>In any case, when deriving lines I've found it pays to re-screen embryos at 5 days, particularly when using mCherry.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-10031587094987751402007-07-31T09:54:00.000-06:002007-07-31T09:54:00.000-06:00Owen--Our understanding (third-hand through Nathan...Owen--<BR/><BR/>Our understanding (third-hand through Nathan Lawson) is that the att sites in pDONR P4-P1R are just especially prone to self-recombination.<BR/><BR/>Obviously with donor vectors there is the problem that they will like to shed the ccdB cassette, so there is strong selective pressure for mutations and/or self-recombination. Careful microbiological technique (using plates poured with chlor/kan) seems to help. Presumably, we have a good colony and make a glycerol stock, there are fewer divisions at which selective pressure can act when we grow up from the glycerol stock.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-43021057192062320462007-07-12T16:55:00.000-06:002007-07-12T16:55:00.000-06:00We are working to prepare pDONR P4-P1R and want to...We are working to prepare pDONR P4-P1R and want to understand what makes a particular prep of the plasmid less likely to undergo self-recombination. Do the att sites readily mutate to facilitate recombination? Kristen has found that a good plasmid - bacteria combination can be frozen and new DNA preps generated from this frozen stock with favorable results. What makes this plasmid - bacterial combination work whereas other combinations lead to recombination?Unknownhttps://www.blogger.com/profile/03869930675308866119noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-73927614120777990512007-06-14T16:46:00.000-06:002007-06-14T16:46:00.000-06:00Margaret--You said "substituting an EcoRI site for...Margaret--<BR/><BR/>You said "substituting an EcoRI site for the 6 amino acids at the end of the IRES and adding another 3 amino acids before the start site of our 3’ element." Did you mean basepairs rather than amino acids? In any case, I would not change any of the basepairs in the IRES sequence, as to my knowledge this may well prevent it from working (I would be happy for a virologist to correct me).<BR/><BR/>My feeling is that you should preserve the entire IRES sequence, including AUG 11, and then keep your 3' element *in frame* with this AUG (as are our EGFP sequences). Spacing shouldn't matter.<BR/><BR/>And no, the polyA site shouldn't be strictly necessary; the polyA site in the destination vector should do the trick.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-79338547665300013672007-06-12T22:10:00.000-06:002007-06-12T22:10:00.000-06:00We’re planning to make a 3’ entry construct with a...We’re planning to make a 3’ entry construct with an IRES sequence, and I have two questions about that. First, how critical is the sequence and/or spacing between the final ATG in the IRES and the ATG that starts the 3’ element? (Our cloning plan involves starting with the IRES sequence from your 3’ IRES-EGFP unit, but substituting an EcoRI site for the 6 amino acids at the end of the IRES and adding another 3 amino acids before the start site of our 3’ element.) And second, all of your 3’ constructs contain polyA sequences. Is this necessary if we’re using the pDestTol2CG2 destination vector (since it has an SV40 late polyA site)? Thank you in advance for any advice!Margarethttps://www.blogger.com/profile/13224799115164409056noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-22720818549984109882007-06-08T09:39:00.000-06:002007-06-08T09:39:00.000-06:00Will --I just want to add to what Chi-Bin said reg...Will --<BR/>I just want to add to what Chi-Bin said regarding the pDONR P4-P1R: there are a couple of things that we've done to make things easier. First, when transforming the plasmid, the bugs must be plated on Kan/Chlor plates. Early on, the lab used Kan plates with some amount of liquid chloramphenicol pipetted on; we believe this caused a lot of problems for us. We think the plates must be poured with both antibiotics mixed in the media. Second, we now have glycerol stocks of plasmids that have worked well before; after streaking these out (on the Kan/Chlor plates), they work very reliably. And yes -- I perform restriction digests and test BPs on all preps. I hope this helps -- good luck!Kristen Kwanhttps://www.blogger.com/profile/03573699538577750070noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-8486268747797875942007-06-08T07:00:00.000-06:002007-06-08T07:00:00.000-06:00Will--it sounds like you have a bad prep of pDONR ...Will--it sounds like you have a bad prep of pDONR P4-P1R. This is a problem that we struggled with early on, as well.<BR/><BR/>For *propagating* pDONR P4-P1R, we follow the Lawson lab protocol: grow up six large cultures (midi- or maxiprep), take a few ml of each to do minipreps and spin down and freeze the bacterial pellet from the rest, then do restriction digests and try test BP reactions with each miniprep; then take the frozen pellet from one of the ones that worked to do a midi- or maxiprep.<BR/><BR/>Is this what you tried? The last time that we tried this, I believe all six preps worked fine. (Kristen, did we do restriction digests *and* test BPs on all six?)<BR/><BR/>The only other thing that I can think of is to perhaps be a bit more careful than usual with your microbiological technique; you don't want to add any selection pressure since this plasmid is slightly unstable.<BR/><BR/>Hope this helps.<BR/><BR/>Chi-BinChi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-31103528716270053232007-06-08T04:26:00.000-06:002007-06-08T04:26:00.000-06:00Dear Chi-BinI have a question concerning self-rec...Dear Chi-Bin<BR/><BR/>I have a question concerning self-recombination of the pDNORp4-P1R plasmid. I have started making entry clones here in the lab - but have the problem that the p4-P1R has a very high rate of self-recombination in my hands. I have already used the Lawson lab protocol to select for non-recombining clones but did not find any clones with no self-recombination. I have now tried a couple of BP reactions using the most promising plasmid prerp, but in nearly all cases I only recover empty clones afterwards.<BR/><BR/>I have managed to successfully make one 5´Entry clone, so I am fairly confident that my primer seqeunces and enzyme mix are OK.<BR/><BR/>Do you have any suggestions as to how I could improve the efficiency for this? Should I re-prep the plasmid and try again?<BR/><BR/>Thanks in advance for your help, and for all the hard work from your lab which has made this techology available to us.<BR/><BR/>WillWill Nortonhttps://www.blogger.com/profile/01228746028214817067noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-10280016097342650852007-06-07T21:09:00.000-06:002007-06-07T21:09:00.000-06:00Thanks for your swift response.Re transposon size,...Thanks for your swift response.<BR/><BR/>Re transposon size, so I don't have to worry about it from now on unless the size of a middle entry clone is over 6 kb. That's good to know.<BR/><BR/>Re EF1a, yes I agree with you that expression driven by EF1a starts to turn off at about 18 hr. Yet some transgenic lines keep glowing without tailing off.<BR/><BR/>I know this from my experience with generating EGFP transgenic fish driven by EF1a; some lines show very bright fluorescence at least until 3 dpf, after which I don't know how bright it would be. I guess it has something to do with position effect.<BR/><BR/>Anyway, like you said, beta-actin promoter seems to be a safe bet. <BR/><BR/>Thanks. SYfishontherunhttps://www.blogger.com/profile/07119307203231171315noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-24903371177601511092007-06-07T06:38:00.000-06:002007-06-07T06:38:00.000-06:00Hi Seok-Yong,Thanks for the kind words. Really mos...Hi Seok-Yong,<BR/><BR/>Thanks for the kind words. Really most of the credit should go to Kristen Kwan, who did most of the work. Also to Ben Mangum and Esther Fujimoto.<BR/><BR/>I went back and looked at the raw sequence for pME-mCherry-CAAX. At the basepair you mention, there is actually a double peak in the chromatogram; one peak gives CAC (correct), and the other gives GAC (incorrect, as you say). So I suspect that the original miniprep that we sequenced was a mixture of two clones; likely the GAC clone has been propagated for the maxiprep that we spotted. We will double-check the sequence and post the corrected version on the wiki.<BR/><BR/>As for beta-actin vs EF1alpha, our experience (matched by others, I think) is that the Xenopus EF1alpha fused enhancer-promoter from Paul Krieg is fine for early expression of zebrafish, but starts to turn off after about 18 hpf (this is what I saw long ago when I made several independent EF1alpha:gfp stable lines). beta-actin seems to stay on even into adulthood.<BR/><BR/>In terms of overall transposon size, both Koichi Kawakami and Steve Ekker's labs have shown that Tol2 tolerates inserts up to at least 10 kb without much problem. We have made multiple stable lines with inserts in the ~9 kb range with very high rates of transgenesis (>30% injected fish transmit the transgene).Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-78184820977360450502007-06-07T01:15:00.000-06:002007-06-07T01:15:00.000-06:00Hi,First off, I'd like to thank Dr. Chien for deve...Hi,<BR/><BR/>First off, I'd like to thank Dr. Chien for developing this wonderful system.<BR/><BR/>I have one comment and one question.<BR/><BR/>I found a mutation in mCherry of pME-mCherryCAAX upon completing multisite gateway cloning (beta-actin + mCherryCAAX + pA). The mutation is H80D (mCherry coordinates (GenBank accession # AY678264; CAC -> GAC). I don't know how this mutation could affect mCherry fluorescence.<BR/><BR/>Re p5E-bactin, is there any difference between 5.2 kb beta-actin promoter and EF1a promoter in terms of expression level and being ubiquitous? The reason I'm asking this question is that I'd like to use a smaller ubiquitous promoter if there's no difference between them. My concern is that big promoter increases the total size of DNA to be integrated into genome, which in turn might reduce the chance of integration. However, I gather there must have been some reason you generated p5E-bactin.<BR/><BR/>Thanks!fishontherunhttps://www.blogger.com/profile/07119307203231171315noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-66187114343290777862007-06-05T10:32:00.000-06:002007-06-05T10:32:00.000-06:00thanks, chi-bin. we could certainly test out the ...thanks, chi-bin. we could certainly test out the IRES mCherry cconstruct for you. we have recently shifted over to using all the fruit fluorescent proteins, and have all the optimal filter sets.<BR/><BR/>Also, I will start woking on making some other IRES constructs. I will keep you posted.Unknownhttps://www.blogger.com/profile/16212727601921614009noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-75721862412823084482007-06-05T10:14:00.000-06:002007-06-05T10:14:00.000-06:00David-As you probably guessed, we did not check fo...David-<BR/>As you probably guessed, we did not check for expression in blood. So I don't know if our 5.3 kb bactin2 fragment is better than your 2.7 kb fragment. I'd be interested to hear the answer...<BR/><BR/>I've just posted some <A HREF="http://chien.neuro.utah.edu:16080/tol2kitwiki/index.php/Sample_results_with_the_Tol2kit#Validation_of_IRES_clones" REL="nofollow">images</A> showing sample results with the IRES clones.<BR/><BR/>Clearly the second cistron is substantially dimmer than the first cistron. We made a set of IRES-mCherry constructs but were not able to see the mCherry, at least on our confocal. It would of course be great to have red IRES constructs, and we'd love to hear suggestions for red FPs to try. (Or even better, for someone else to make the 3' clones!)Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-62015679608098369052007-06-05T08:55:00.001-06:002007-06-05T08:55:00.001-06:00I was wondering if there was any way you had creat...I was wondering if there was any way you had created more IRES constructs with different fluorophores (such as IRES-mCherry, IRES-AmCyan, IRES-mOrange, or IRES-dsRed).Unknownhttps://www.blogger.com/profile/16212727601921614009noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-246362344545918592007-06-05T08:55:00.000-06:002007-06-05T08:55:00.000-06:00The entry clone p5E-bactin2: Is the bactin expres...The entry clone p5E-bactin2: Is the bactin expression really ubiquitous? We want to mark blood cells, and we previously tried a shorter version of this promoter (2.7kb). It doesn't seem to work. However, when we moved up to using 10kb we see expression in the blood. I was just wondering if you see expression in blood cells, or if you have ever really looked. If not, it is an easy experiment for me to perform. The paper listed as a reference doesn't answer this question- it simply says that expression is ubiquitous.Unknownhttps://www.blogger.com/profile/16212727601921614009noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-21709054499792526652007-06-04T15:32:00.000-06:002007-06-04T15:32:00.000-06:00Hi Andreas,I just double-checked the posted sequen...Hi Andreas,<BR/>I just double-checked the posted sequence; it's correct. Perhaps you didn't import the sequence correctly? Note that this is a FASTA file with multiple fragments, including the sequences of various components of the clone. If you imported them all as though they were a single sequence, that would explain the duplications you saw.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-23776292723518993012007-06-04T15:18:00.000-06:002007-06-04T15:18:00.000-06:00Dear Chi-Bin,thanks for the prompt reply. It is h...Dear Chi-Bin,<BR/><BR/>thanks for the prompt reply. It is helpful to know which sites can be used. I just want to point out that the sequence that I can access from your page is still wrong. It has the entire Bluescript MCS repeated 3 times (pos 718 - 823; 3068 - 3173; 3253 - 3358). Thus, I have e.g. 3 Hind III sites predicted. I'll work with the sites you indicated. Thanks,<BR/>AndreasUnknownhttps://www.blogger.com/profile/07444504894326127397noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-7507993386145269122007-06-04T14:54:00.000-06:002007-06-04T14:54:00.000-06:00Dear Andreas,Unfortunately, you're wrong in this a...Dear Andreas,<BR/><BR/>Unfortunately, you're wrong in this assumption. The posted sequence is correct. The problem is that the pDONR backbone has a lot of restriction sites. I guess Invitrogen didn't make them friendly because they expected everyone to clone in by BP reaction. All that we did was to add the pBS MCS (from T7 to T3 primers).<BR/><BR/>However, there are still some useful sites:<BR/><BR/>KpnI, DraII, XhoI, SalI, HindIII, SmaI, BamHI, SpeI, SacII are all unique.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-59254879726704348842007-06-04T10:52:00.000-06:002007-06-04T10:52:00.000-06:00I have a question regarding the p5E-MCS vector. I...I have a question regarding the p5E-MCS vector. I wanted to use it to clone a promoter fragment into it by standard restriction cloning. I downloaded the posted sequence and it seems to repeat most of the MCS just after the attR1 site. If I click on "unique" sites in my DNA analysis program, there are virtually none left. I assume there is some error somewhere and that the Bluescript MCS sites are actually unique and can be used for cloning? Or am I wrong in this assumption? Thanks,<BR/><BR/>AndreasUnknownhttps://www.blogger.com/profile/07444504894326127397noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-81121998765935212702007-05-12T21:33:00.000-06:002007-05-12T21:33:00.000-06:00Arul--yes, as our protocol says, you need highly c...Arul--yes, as our protocol says, you need highly competent cells like OneShot Top10 for the LR reactions.<BR/><BR/>I think you need to do some positive controls (e.g. beta-actin-gfp-pA).Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.com