From ucgahar from ucl.ac.uk Fri May 2 04:20:04 2008 From: ucgahar from ucl.ac.uk (carole wilson) Date: Fri May 2 11:26:36 2008 Subject: [Zbrafish] Adult brinshrimp Message-ID: <161045C8-9640-41FC-B926-DAB1248C41D0@ucl.ac.uk> Dear all, Does anyone feed live adult brineshrimp to adult zebrafish? Thanks Carole Carole Wilson Fish Facility Manager University College London ucgahar@ucl.ac.uk -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080502/0b2197fa/attachment.html From david from zebrafish.org Fri May 2 12:44:39 2008 From: david from zebrafish.org (David Lains) Date: Fri May 2 12:50:40 2008 Subject: [Zbrafish] Adult brinshrimp In-Reply-To: <161045C8-9640-41FC-B926-DAB1248C41D0@ucl.ac.uk> References: <161045C8-9640-41FC-B926-DAB1248C41D0@ucl.ac.uk> Message-ID: <056601c8ac7c$351e9ce0$9f5bd6a0$@org> Hi Carole We do not. The adults have considerably more shell to meat then the nauplii. The newly hatched bs are much higher in fats and proteins, easier to produce and do not pose a bio security risk. Best Fishes David Lains <}}}>< Aquaculturist, Research Assistant Zebrafish International Resource Center 5274 University of Oregon Eugene, Or 97403 Email: david@zebrafish.org pH: (541) 346-6028 ext. 18 fax: (541) 346-6151 From: zbrafish-bounces@oat.bio.indiana.edu [mailto:zbrafish-bounces@oat.bio.indiana.edu] On Behalf Of carole wilson Sent: Friday, May 02, 2008 2:20 AM To: bionet-organisms-zebrafish@moderators.isc.org Subject: [Zbrafish] Adult brinshrimp Dear all, Does anyone feed live adult brineshrimp to adult zebrafish? Thanks Carole Carole Wilson Fish Facility Manager University College London ucgahar@ucl.ac.uk -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080502/6adde15d/attachment.html From zhangch from ohsu.edu Fri May 2 15:42:24 2008 From: zhangch from ohsu.edu (Chao Zhang) Date: Fri May 2 15:44:30 2008 Subject: [Zbrafish] Larval zebrafish melanocyte aggregation Message-ID: WT larval zebrafish keeps melanocytes dispersed until 4dpf. Then they will aggregate between 4 and 5dpf. So what kind of physiological factors contribute to this process. The balance of a-MSH/MCH, etc? What kind of reasons could delay this normal process(Dispersed melanophore remains until 5 or 6dpf and aggregate since 7dpf)? Chao From jason.cockington from gmail.com Sun May 4 20:11:04 2008 From: jason.cockington from gmail.com (Leviathan) Date: Mon May 5 10:06:57 2008 Subject: [Zbrafish] Re: Adult brinshrimp References: <161045C8-9640-41FC-B926-DAB1248C41D0@ucl.ac.uk> Message-ID: <0aa05a14-b0d0-4619-8503-3de98a8020e8@a9g2000prl.googlegroups.com> Hi Carole, We don't either. We feed 48hr naupili to fry and adult alike. Although we have pondered the idea of growing algae, and closing the life cycle in our facility. But at this stage, and the foreseeable future, we will just use the 48s. Jason From sjohnson from genetics.utah.edu Mon May 5 10:31:26 2008 From: sjohnson from genetics.utah.edu (Sharon Johnson) Date: Mon May 5 10:42:16 2008 Subject: [Zbrafish] Decapsulated dehydrated Brine Shrimp Message-ID: Has anyone tried using commercially decapsulated/dehydrated brine shrimp nauplii in place of 24-48hr post-hatch brine shrimp? Even wroking within sight of the Great Salt Lake, we are having difficulties obtaining good quality artemia cysts. We are doing some experiments with a product from INVE Aquaculture (not an endorsement of this company) that costs about ? of the artemia cysts by weight and is 100% usable. Thanks - Sharon Sharon E. Johnson Senior Lab Specialist University of Utah Zebrafish Core Facility 30 N. 1900 East 5C 124 SOM Salt Lake City, Utah 84132 Phone: (801) 585-6574 Fax: (801) 585-6364 -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080505/e17e5102/attachment.html From david from zebrafish.org Mon May 5 12:17:29 2008 From: david from zebrafish.org (David Lains) Date: Mon May 5 12:24:50 2008 Subject: [Zbrafish] Re: Adult brinshrimp In-Reply-To: <0aa05a14-b0d0-4619-8503-3de98a8020e8@a9g2000prl.googlegroups.com> References: <161045C8-9640-41FC-B926-DAB1248C41D0@ucl.ac.uk> <0aa05a14-b0d0-4619-8503-3de98a8020e8@a9g2000prl.googlegroups.com> Message-ID: <01d201c8aed3$e8e0acb0$baa20610$@org> Hi Jason Why 48 hr nauplii? Are you gut loading them? You might try Reed Mariculture's algae products. They are very easy to use and can be kept in the freezer. Best Fishes David Lains <}}}>< Aquaculturist, Research Assistant Zebrafish International Resource Center 5274 University of Oregon Eugene, Or 97403 Email: david@zebrafish.org pH: (541) 346-6028 ext. 18 fax: (541) 346-6151 -----Original Message----- From: zbrafish-bounces@oat.bio.indiana.edu [mailto:zbrafish-bounces@oat.bio.indiana.edu] On Behalf Of Leviathan Sent: Sunday, May 04, 2008 6:11 PM To: bionet-organisms-zebrafish@moderators.isc.org Subject: [Zbrafish] Re: Adult brinshrimp Hi Carole, We don't either. We feed 48hr naupili to fry and adult alike. Although we have pondered the idea of growing algae, and closing the life cycle in our facility. But at this stage, and the foreseeable future, we will just use the 48s. Jason _______________________________________________ Zbrafish mailing list Zbrafish@net.bio.net http://www.bio.net/biomail/listinfo/zbrafish From claudia_hohn from hotmail.com Tue May 6 11:18:56 2008 From: claudia_hohn from hotmail.com (Claudia) Date: Tue May 6 11:20:11 2008 Subject: [Zbrafish] Re: Decapsulated dehydrated Brine Shrimp References: Message-ID: <6537884c-0786-4dce-9c63-9517d13182e5@m73g2000hsh.googlegroups.com> On May 5, 10:31 am, Sharon Johnson wrote: > Has anyone tried using commercially decapsulated/dehydrated brine shrimp nauplii in place of 24-48hr post-hatch brine shrimp? Even wroking within sight of the Great Salt Lake, we are having difficulties obtaining good quality artemia cysts. We are doing some experiments with a product from INVE Aquaculture (not an endorsement of this company) that costs about ? of the artemia cysts by weight and is 100% usable. Thanks - Sharon > > Sharon E. Johnson > Senior Lab Specialist > University of Utah > Zebrafish Core Facility > 30 N. 1900 East > 5C 124 SOM > Salt Lake City, Utah 84132 > Phone: (801) 585-6574 > Fax: (801) 585-6364 Hi, we are using decapsulated brine shrimp as a starting feed for commercial food. Starting on day 16 pf once we stop feeding paramecium we start the shellfree artemia (www.artemia-international.com) in addition to 24h artemia nauplii (www.artemia-international.com) until about 22 dpf, then we start feeding Golden Pearl feed (200-300um) (www.artemia-international.com)...... The shellfree artemia is very very small and I do not think it is suitable for adult fish. Claudia Hohn, Ph.D. PO Box 3088 Mississippi State University MS, 39762 USA From mwinandy from ulg.ac.be Wed May 7 02:38:53 2008 From: mwinandy from ulg.ac.be (Marie Winandy) Date: Wed May 7 14:35:33 2008 Subject: [Zbrafish] artemia cysts Message-ID: <200805070938530687.0011EB09@smtp.ulg.ac.be> Dear Sharon, Here we use also INVE cysts. We allow them to hatch at 28?C with light, in a 24h delay. But before putting themin the hatchery, we soften them by incubating 30' in RO water and then 5' in NaClO. After that we rinse thoroughly and then put in the hatchery with salted water. This NaClO treatment allows a rapid hatching and the cycsts are soft, and then don't hurt the gut of the fish. We use the same artemias for fries and adults (since day 9, increasing the given amount). Hope it helps, best regards, Marie Winandy, PhD Universit? de Li?ge GIGA B34 - Zebrafish Platform Avenue de l'H?pital 1 B-4000 Li?ge - Sart Tilman Belgique T?l: +32.4.366.99.71 +32.4.366.33.38 +32.476.97.25.33 Fax: +32.4.366.41.98 email : mwinandy@ulg.ac.be or zebrafish.giga@ulg.ac.be http://www.giga.ulg.ac.be/extranet/services.htm -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080507/61101750/attachment.html From dolores.bonaparte from gmail.com Thu May 8 02:56:58 2008 From: dolores.bonaparte from gmail.com (Doloresbf) Date: Thu May 8 10:53:35 2008 Subject: [Zbrafish] drug dosage and administration Message-ID: Hello, I have one researcher who needs to administer dexamethasone and 4-OHT to adult ZFish, to induce tgn expression. Do you know where I can find dosages and other indications concerning this? I'm new to the Zebra world... :o) Thanks in advance. Dolores ----------------------------------------- Dolores Bonaparte, DVM Instituto de Medicina Molecular Animal Facility Portugal Ph:+351 21 799 9550 Fax: +351 21 799 9459 email: doloresbf@fm.ul.pt From pinaleno from gmail.com Tue May 13 08:36:47 2008 From: pinaleno from gmail.com (Chris) Date: Tue May 13 11:03:07 2008 Subject: [Zbrafish] Need for Cryogenic Services? Message-ID: <2f4c49e1-e418-46a5-abde-75e53a263518@m45g2000hsb.googlegroups.com> Hi All: As you know, one of the myriad challenges in managing a zebrafish facility involves "backing up" important strains as frozen sperm, both to cut down on the number of lines that have to be maintained within a facility as "swimming copies" and to aid in the recovery of a research program in the event of a disaster. Presently, ZIRC is only the answer IF you have a line that you are willing to share with the scientific community, and IF the line is deemed important enough scientifically to merit storing for cataloguing and subsequent distribution. For all other applications, you are on your own. I am aware that a number of facilities (although I don't know if it is the majority or minority) cryopreserve sperm of important lines. This is a time-consuming and difficult process - to do it properly, at least - especially with limited resources. My suspicion is that most facilities, if they are doing it at all, probably aren't doing it very well. I personally have been involved in a laboratory where a "resource" of frozen stock was actually not a resource at all, but rather a vat full of non-viable sperm. There were a number of reasons for this, but it was mostly due to lack of resources to oversee this operation properly. I suspect that there are many such "resources" out there in the community…zebrafish "binkies", if you will. My question to the community is whether or not zebrafish users would utilize a professional service, similar to what is currently available for rodents, IF such a resource (reliable and demonstrated) existed. Why or why not? Thanks in advance for your time, Chris Christian Lawrence Children’s Hospital Boston Enders/ARCH/Aquatics 320 Longwood Avenue Boston, MA 02115 617.919.2738 (office) 617.730-0836 (fax) christian.lawrence@childrens.harvard.edu From busquet.francois from gmail.com Wed May 14 08:30:37 2008 From: busquet.francois from gmail.com (=?ISO-8859-1?Q?fran=E7ois?=) Date: Wed May 14 12:35:46 2008 Subject: [Zbrafish] Measurement of compound uptake into the fish embryo Message-ID: <0e26645e-8da3-4a5a-86d3-ec49eb4eb801@r66g2000hsg.googlegroups.com> Hi, I am actually reviewing possible methods to measure compound uptake into the fish embryos. Until now, LC-MS protocols are available. However, I also heard about algorithms but I could not find any reference. I would be grateful if anybody knew about it or others methods. Regards, fran?ois From chris.cyk from gmail.com Tue May 13 20:29:20 2008 From: chris.cyk from gmail.com (chris.cyk@gmail.com) Date: Wed May 14 12:36:06 2008 Subject: [Zbrafish] IP3R Antibody for Zebrafish Message-ID: <5cabaeb9-b680-4dd1-a0e8-bd3828bc8595@u12g2000prd.googlegroups.com> I'm looking for the antibody that labels inositol 1,4,5-trisphosphate receptors (IP3R) in zebrafish. I've tried several commerical available antibodies, but they're all not working. Do you know any IP3R antibody that is working in zebrafish? Thank you very much. Chris Cheung Graduate student Department of Biology The Hong Kong University of Science & Technology Clear Water Bay, Kowloon, Hong Kong Tel: (852) 2358-7324 Fax: (852) 2358-7323 From chris.cyk from gmail.com Tue May 13 20:50:57 2008 From: chris.cyk from gmail.com (Chris) Date: Wed May 14 12:36:27 2008 Subject: [Zbrafish] IP3R Antibody for Zebrafish Message-ID: I'm looking for the antibody that labels inositol 1,4,5-trisphosphate receptors (IP3R) in zebrafish. I've tried several commerical available antibodies, but they're all not working. Do you know any IP3R antibody that is working in zebrafish? Thank you very much. Chris Cheung Graduate student Department of Biology The Hong Kong University of Science & Technology Clear Water Bay, Kowloon, Hong Kong Tel: (852) 2358-7324 Fax: (852) 2358-7323 From jyyoungjimmy from gmail.com Thu May 15 12:55:09 2008 From: jyyoungjimmy from gmail.com (Jim Young) Date: Thu May 15 13:04:59 2008 Subject: [Zbrafish] Re: IP3R Antibody for Zebrafish Message-ID: <57f211620805151055n76a66813h90269640634c4a4b@mail.gmail.com> You can find some good antibodies from here: http://www.genscript.com/cgi-bin/products/rec_antibody.cgi On Thu, May 15, 2008 at 1:03 PM, wrote: > Send Zbrafish mailing list submissions to > zbrafish@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/zbrafish > or, via email, send a message with subject or body 'help' to > zbrafish-request@net.bio.net > > You can reach the person managing the list at > zbrafish-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Zbrafish digest..." > > > Today's Topics: > > 1. Measurement of compound uptake into the fish embryo (fran?ois) > 2. IP3R Antibody for Zebrafish (chris.cyk@gmail.com) > 3. IP3R Antibody for Zebrafish (Chris) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 14 May 2008 06:30:37 -0700 (PDT) > From: fran?ois > Subject: [Zbrafish] Measurement of compound uptake into the fish > embryo > To: bionet-organisms-zebrafish@moderators.isc.org > Message-ID: > <0e26645e-8da3-4a5a-86d3-ec49eb4eb801@r66g2000hsg.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > I am actually reviewing possible methods to measure compound uptake > into the fish embryos. Until now, LC-MS protocols are available. > However, I also heard about algorithms but I could not find any > reference. > I would be grateful if anybody knew about it or others methods. > Regards, > fran?ois > > > > ------------------------------ > > Message: 2 > Date: Tue, 13 May 2008 18:29:20 -0700 (PDT) > From: chris.cyk@gmail.com > Subject: [Zbrafish] IP3R Antibody for Zebrafish > To: bionet-organisms-zebrafish@moderators.isc.org > Message-ID: > <5cabaeb9-b680-4dd1-a0e8-bd3828bc8595@u12g2000prd.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > I'm looking for the antibody that labels inositol 1,4,5-trisphosphate > receptors (IP3R) in zebrafish. I've tried several commerical available > antibodies, but they're all not working. Do you know any IP3R antibody > that is working in zebrafish? Thank you very much. > > Chris Cheung > Graduate student > Department of Biology > The Hong Kong University of Science & Technology > Clear Water Bay, Kowloon, Hong Kong > Tel: (852) 2358-7324 > Fax: (852) 2358-7323 > > > > > ------------------------------ > > Message: 3 > Date: Tue, 13 May 2008 18:50:57 -0700 (PDT) > From: Chris > Subject: [Zbrafish] IP3R Antibody for Zebrafish > To: bionet-organisms-zebrafish@moderators.isc.org > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > I'm looking for the antibody that labels inositol 1,4,5-trisphosphate > receptors (IP3R) in zebrafish. I've tried several commerical available > antibodies, but they're all not working. Do you know any IP3R antibody > that is working in zebrafish? Thank you very much. > > Chris Cheung > Graduate student > Department of Biology > The Hong Kong University of Science & Technology > Clear Water Bay, Kowloon, Hong Kong > Tel: (852) 2358-7324 > Fax: (852) 2358-7323 > > > > ------------------------------ > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > > End of Zbrafish Digest, Vol 36, Issue 7 > *************************************** > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080515/754d37fb/attachment.html From waldrons from u.washington.edu Tue May 20 14:20:39 2008 From: waldrons from u.washington.edu (Steven Waldron) Date: Thu May 22 11:17:56 2008 Subject: [Zbrafish] manipulating micromanipulators Message-ID: <48332487.80103@u.washington.edu> Hello, I have a couple of workhorse Narishige M-152 micromanipulators that need some maintenance- the X-axis knob needs tightening. Does anyone know how to do this? best, Steve From Ian.Bessell from zeiglerfeed.com Thu May 22 08:22:35 2008 From: Ian.Bessell from zeiglerfeed.com (IBessell) Date: Thu May 22 11:27:02 2008 Subject: [Zbrafish] Zeigler Bros Feed - New Member to Group Message-ID: <5844d567-5907-4d00-be45-58935f068b49@x35g2000hsb.googlegroups.com> Hello Zebrafish Community: I am a new member to this group and also an industry partner as an employee of Zeigler Bros Feed. We currently manufacture a variety of aquatic diets for both the aquaculture industry and Aquatic Lab Industry including larval fish diets and an Adult Zebrafish Diet. Please visit our website www.zeiglerfeed.com or e-mail me directly if myself or Zeigler Bros can help with anything related to Zebrafish nutrition and/or foods. Looking forward to working with you all Ian ________________________________________ Ian S. Bessell, M.A.B. Sales Specialist-Pet, Zoo and Lab Diets Zeigler Bros.Feed, Inc. 717-968-6915 cell 786-242-7157 Florida office 786-242-6549 fax Skype Contact: ian.bessell Ian.Bessell@zeiglerfeed.com www.zeiglerfeed.com www.monsterdiets.com From gilbert.weidinger from biotec.tu-dresden.de Fri May 23 10:54:13 2008 From: gilbert.weidinger from biotec.tu-dresden.de (Gilbert Weidinger) Date: Tue May 27 11:21:22 2008 Subject: [Zbrafish] morpholino blues Message-ID: <4836E8A5.3070703@biotec.tu-dresden.de> hi all, I wonder whether it's conceivable that some genes cannot be targeted with morpholinos. In many years of using MOs I have come to accept that only about 50% of them work, but now we have a case in the lab where NONE out of 4 MOs targeting the same gene has any effect. We have 2 splice blockers: they do not alter splicing as determined by RT-PCR. We have 2 translation blockers: they do not inhibit translation of RNA reporter constructs (5'UTR fused to cherry). We inject up to 10?g/?l and use both the RT-PCR assay and the reporter RNAs successfully for other MOs (against other genes). Genetools claims that 70% of all MOs knock down their target... Of note is that all 4 MOs are tagged with fluorescein. We don't have a lot of experience with these. Is it possible that they are less efficient than untagged MOs? I'd appreciate getting some feedback on whether there are other stories like this out there or whether we are simply exceptionally unlucky. thanks gilbert ----------------------------------------------------------- Gilbert Weidinger, PhD Group Leader SFB 655 Biotechnology Center & Center for Regenerative Therapies Technical University of Dresden Mail address: Biotechnology Center Tatzberg 47-51 01307 Dresden Germany Tel. office: 0049 351 463 40120 Tel. lab: 0049 351 463 40108 Tel. secretary: 0049 351 463 40345 Fax: 0049 351 463 40348 email: gilbert.weidinger@biotec.tu-dresden.de web: http://www.biotec.tu-dresden.de/weidinger From LiuZijuan from gmail.com Sun May 25 16:26:49 2008 From: LiuZijuan from gmail.com (detroitriver) Date: Tue May 27 11:22:16 2008 Subject: [Zbrafish] Re: Measurement of compound uptake into the fish embryo References: Message-ID: On May 14, 9:30?am, fran?ois wrote: > Hi, > I am actually reviewing possible methods to measure compound uptake > into the fish embryos. Until now, LC-MS protocols are available. > However, I also heard about algorithms but I could not find any > reference. > I would be grateful if anybody knew about it or others methods. > Regards, > fran?ois I used ICP-MS to measure metalloid-it is very accurate equipment, depends on if it is available on-site. From rburdine from Princeton.EDU Tue May 27 14:30:24 2008 From: rburdine from Princeton.EDU (Burdine, Rebecca D) Date: Tue May 27 14:37:29 2008 Subject: [Zbrafish] morpholino blues In-Reply-To: <4836E8A5.3070703@biotec.tu-dresden.de> References: <4836E8A5.3070703@biotec.tu-dresden.de> Message-ID: <04AF14C4EF3CF34682460FB1FDBA98C01B743E@MBCLUSTER.pu.win.princeton.edu> Hi Gilbert, In general I would say 50% or less of the MOs we order work. I know they don't work because most of the time we have actual mutants we cannot phenocopy. We want the MOs so we can have all the embryos essentially mutant for biochemistry and other experiments. When we order MOs to knock down genes where we have no idea what the phenotype is, I am always very skeptical. Some odd results for us include: One case where we have a splice morpholino we are 99% sure actually affects maternal. In another case we have ordered 5 different MO to a gene (3 start site, 2 splice) and only 1 splice MO works sort of. At least twice we have ordered MOs from publications and can't get them to work either. When we contact the authors we are told they only work in TL or AB or that we have to really titrate or do something else. My feeling from this is lots of people have similar issues. When they work, they are phenomenal. We have some that give great results at 250pg per embryo! When they don't, I mourn my $400+. 8) I always have Genetools design mine for us so I really believe they are as good as we can predict. However for so many of our genes we don't have a great handle on the 5' end or alternative messages so maybe this is part of the problem. I'd be interested in hearing other people's experiences too. Becky --------------------------------------------------- Rebecca D. Burdine, Ph.D. Assistant Professor Dept. of Molecular Biology Princeton University Washington Road Mof 433 Princeton, NJ 08544 Phone: (609) 258-7515 Fax: (609) 258-1343 Email: rburdine@princeton.edu Admin Assistant: Cathy Falk (609) 258-1604 > -----Original Message----- > From: zbrafish-bounces@oat.bio.indiana.edu > [mailto:zbrafish-bounces@oat.bio.indiana.edu] On Behalf Of > Gilbert Weidinger > Sent: Friday, May 23, 2008 11:54 AM > To: Zbrafish@magpie.bio.indiana.edu > Subject: [Zbrafish] morpholino blues > > hi all, > > I wonder whether it's conceivable that some genes cannot be > targeted with morpholinos. In many years of using MOs I have > come to accept that only about 50% of them work, but now we > have a case in the lab where NONE out of 4 MOs targeting the > same gene has any effect. > > We have 2 splice blockers: they do not alter splicing as > determined by RT-PCR. > We have 2 translation blockers: they do not inhibit > translation of RNA reporter constructs (5'UTR fused to cherry). > > We inject up to 10?g/?l and use both the RT-PCR assay and the > reporter RNAs successfully for other MOs (against other > genes). Genetools claims that 70% of all MOs knock down their > target... > > Of note is that all 4 MOs are tagged with fluorescein. We > don't have a lot of experience with these. Is it possible > that they are less efficient than untagged MOs? > > I'd appreciate getting some feedback on whether there are > other stories like this out there or whether we are simply > exceptionally unlucky. > > thanks > gilbert > > ----------------------------------------------------------- > Gilbert Weidinger, PhD > > Group Leader SFB 655 > Biotechnology Center & Center for Regenerative Therapies > Technical University of Dresden > > Mail address: > > Biotechnology Center > Tatzberg 47-51 > 01307 Dresden > Germany > > Tel. office: 0049 351 463 40120 > Tel. lab: 0049 351 463 40108 > Tel. secretary: 0049 351 463 40345 > Fax: 0049 351 463 40348 > email: gilbert.weidinger@biotec.tu-dresden.de > web: http://www.biotec.tu-dresden.de/weidinger > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > From pavlina.haramis from gmail.com Wed May 28 03:50:43 2008 From: pavlina.haramis from gmail.com (pavlina.haramis@gmail.com) Date: Wed May 28 11:17:35 2008 Subject: [Zbrafish] Re: morpholino blues References: <4836E8A5.3070703@biotec.tu-dresden.de> Message-ID: <6051652c-6f15-4f8d-b387-f8800a0a3c40@26g2000hsk.googlegroups.com> On May 27, 9:30?pm, "Burdine, Rebecca D" wrote: > Hi Gilbert, > > In general I would say 50% or less of the MOs we order work. ?I know they don't work because most of the time we have actual mutants we cannot phenocopy. We want the MOs so we can have all the embryos essentially mutant for biochemistry and other experiments. ?When we order MOs to knock down genes where we have no idea what the phenotype is, I am always very skeptical. > > Some odd results for us include: > One case where we have a splice morpholino we are 99% sure actually affects maternal. ?In another case we have ordered 5 different MO to a gene (3 start site, 2 splice) and only 1 splice MO works sort of. ?At least twice we have ordered MOs from publications and can't get them to work either. ?When we contact the authors we are told they only work in TL or AB or that we have to really titrate or do something else. ?My feeling from this is lots of people have similar issues. > > When they work, they are phenomenal. We have some that give great results at 250pg per embryo! When they don't, I mourn my $400+. 8) > > I always have Genetools design mine for us so I really believe they are as good as we can predict. However for so many of our genes we don't have a great handle on the 5' end or alternative messages so maybe this is part of the problem. > > I'd be interested in hearing other people's experiences too. > > Becky > > --------------------------------------------------- > Rebecca D. Burdine, Ph.D. > Assistant Professor > Dept. of Molecular Biology > Princeton University > Washington Road Mof 433 > Princeton, NJ 08544 > > Phone: (609) 258-7515 > Fax: (609) 258-1343 > Email: rburd...@princeton.edu > Admin Assistant: Cathy Falk (609) 258-1604 > > > -----Original Message----- > > From: zbrafish-boun...@oat.bio.indiana.edu > > [mailto:zbrafish-boun...@oat.bio.indiana.edu] On Behalf Of > > Gilbert Weidinger > > Sent: Friday, May 23, 2008 11:54 AM > > To: Zbraf...@magpie.bio.indiana.edu > > Subject: [Zbrafish] morpholino blues > > > hi all, > > > I wonder whether it's conceivable that some genes cannot be > > targeted with morpholinos. In many years of using MOs I have > > come to accept that only about 50% of them work, but now we > > have a case in the lab where NONE out of 4 MOs targeting the > > same gene has any effect. > > > We have 2 splice blockers: they do not alter splicing as > > determined by RT-PCR. > > We have 2 translation blockers: they do not inhibit > > translation of RNA reporter constructs (5'UTR fused to cherry). > > > We inject up to 10?g/?l and use both the RT-PCR assay and the > > reporter RNAs successfully for other MOs (against other > > genes). Genetools claims that 70% of all MOs knock down their > > target... > > > Of note is that all 4 MOs are tagged with fluorescein. We > > don't have a lot of experience with these. Is it possible > > that they are less efficient than untagged MOs? > > > I'd appreciate getting some feedback on whether there are > > other stories like this out there or whether we are simply > > exceptionally unlucky. > > > thanks > > gilbert > > > ----------------------------------------------------------- > > Gilbert Weidinger, PhD > > > Group Leader SFB 655 > > Biotechnology Center & Center for Regenerative Therapies > > Technical University of Dresden > > > Mail address: > > > Biotechnology Center > > Tatzberg 47-51 > > 01307 Dresden > > Germany > > > Tel. office: ? ?0049 351 463 40120 > > Tel. lab: ? ? ? 0049 351 463 40108 > > Tel. secretary: 0049 351 463 40345 > > Fax: ? ? ? ? ? ?0049 351 463 40348 > > email: ? ? ? ? ?gilbert.weidin...@biotec.tu-dresden.de > > web: ? ? ? ? ? ? ?http://www.biotec.tu-dresden.de/weidinger > > > _______________________________________________ > > Zbrafish mailing list > > Zbraf...@net.bio.net > >http://www.bio.net/biomail/listinfo/zbrafish Hi Gilbert, Becky, We too have had some crazy problems with Morpholinos. We also have a gene that we have 4 MOs against 2 ATG-blocking, 2 splice blocking One splice-blocking works we can show that on RT PCR ( not a massive efficiency though 50-50 wt message, wrongly spliced message) and gives us a phenotype. The ATG-blocking gives us a totally different phenotype! The rescue does not work and the other two MOs give us normal embryos ( probably don't work) We also have a case that we can not phenocopy the mutant by MOs in the same gene. But we also have a case that when you inject the MO in hets, then you have the phenotype ( probably maternal protein?) And the weirdest is that we have 3 MOs against the same gene, the all give the same phenotype, but we can not show molecularly that they work ( the splice-blocking does not block splicing... and the rescue does not work either..... Actually since we are on this "misery-sharing" thread, i have a question since almost none of our rescues work. Do people just clone their gene in the pCS2 or do you put stuff like Kozak sequence etc. Actually could it be that by adding a Kozak you make things worse in terms of expresion?? I know it sounds daft but i am getting desperate. We do have the SV40 polyA etc and obviously we only try to rescue splice-blocking MO- induced phenotypes whenthere are is only 5 bp overlap between the gene and the exogenous mRNA. That was such a cathartic experience! Thanks Becky! From maripelleri from hotmail.com Wed May 28 08:25:54 2008 From: maripelleri from hotmail.com (maria chiara pelleri) Date: Wed May 28 12:22:44 2008 Subject: [Zbrafish] blood extraction Message-ID: Hi! I'm searching the best method for extracting the blood from individual zebrafish. If you had any advice or particular methods for extracting the blood from zf, please, answer me!!! Thanks Maria Chiara Pelleri _________________________________________________________________ Una cena tra amici? Cerca il ristorante con Live Search Maps http://maps.live.it -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080528/9b2f7eca/attachment.html From maripelleri from hotmail.com Thu May 29 06:00:37 2008 From: maripelleri from hotmail.com (maria chiara pelleri) Date: Thu May 29 10:56:33 2008 Subject: FW: [Zbrafish] blood extraction In-Reply-To: References: Message-ID: From: maripelleri@hotmail.com To: zbrafish@magpie.bio.indiana.edu Subject: RE: [Zbrafish] blood extraction Date: Thu, 29 May 2008 09:16:34 +0000 Sorry! I didn't specify that I need to extract the greatest quantity of blood possible because I want to extract the RNA from the sample of blood, I usually kill the fish and I use adult fish. Can you help me? Thanks Maria Chiara Maria Chiara Pelleri University of Bologna, Italy From: maripelleri@hotmail.com To: zbrafish@magpie.bio.indiana.edu Date: Wed, 28 May 2008 13:25:54 +0000 CC: Subject: [Zbrafish] blood extraction Hi! I'm searching the best method for extracting the blood from individual zebrafish. If you had any advice or particular methods for extracting the blood from zf, please, answer me!!! Thanks Maria Chiara Pelleri Windows Live Mail Controlla i tuoi account di posta con un unico programma, ? GRATIS! Windows Live Mail Controlla i tuoi account di posta con un unico programma, ? GRATIS! _________________________________________________________________ Scarica Windows Live, un mondo di programmi per te! http://get.live.com/ -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080529/9788a4bc/attachment.html From claudia_hohn from hotmail.com Fri May 30 11:42:26 2008 From: claudia_hohn from hotmail.com (Claudia) Date: Fri May 30 11:54:57 2008 Subject: FW: [Zbrafish] blood extraction References: Message-ID: <1046af63-fedd-4faf-888f-f7b2457d35a3@b1g2000hsg.googlegroups.com> On May 29, 6:00 am, maria chiara pelleri wrote: > From: maripell...@hotmail.com > To: zbraf...@magpie.bio.indiana.edu > Subject: RE: [Zbrafish] blood extraction > Date: Thu, 29 May 2008 09:16:34 +0000 > > Sorry! > I didn't specify that I need to extract the greatest quantity of blood possible because I want to extract the RNA from the sample of blood, I usually kill the fish and I use adult fish. > Can you help me? > Thanks > Maria Chiara > > Maria Chiara Pelleri > University of Bologna, Italy > > From: maripell...@hotmail.com > To: zbraf...@magpie.bio.indiana.edu > Date: Wed, 28 May 2008 13:25:54 +0000 > CC: > Subject: [Zbrafish] blood extraction > > Hi! > I'm searching the best method for extracting the blood from > individual zebrafish. If you had any advice or particular methods for > extracting the blood from zf, please, answer me!!! > Thanks > Maria Chiara Pelleri > > Windows Live Mail Controlla i tuoi account di posta con un unico programma, ? GRATIS! > > Windows Live Mail Controlla i tuoi account di posta con un unico programma, ? GRATIS! > > _________________________________________________________________ > Scarica Windows Live, un mondo di programmi per te!http://get.live.com/ Hi Maria, we use the method described by P. JAGADEESWARAN, J. P. SHEEHAN, F. E. CRAIG AND D. TROYER: Identification and characterization of zebrafish thrombocytes. British Journal of Haematology, 1999, 107, 731-738. Depending on the size of the zebrafish you get between 5-10ul of blood. Claudia From Lindsey.McFarlane from csiro.au Sat May 31 02:11:18 2008 From: Lindsey.McFarlane from csiro.au (Lindsey.McFarlane@csiro.au) Date: Mon Jun 2 11:14:36 2008 Subject: [Zbrafish] In vitro translation in fish cell lysates Message-ID: <7EDC0EE19405664899D1647A6C9FF6D91DD9DC@extas4-hba.tas.csiro.au> Gday - does anyone know of a protocol for in vitro translation in fish cell lysates Cheers Lindsey -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080531/0ddc5fdc/attachment.html