From xiaw from umbi.umd.edu Mon Nov 2 19:19:08 2009 From: xiaw from umbi.umd.edu (Wei Xia) Date: Mon Nov 2 19:21:02 2009 Subject: [Zbrafish] Red fluorescent reporters for transgenic fish Message-ID: <4C727143-31AF-4701-9DC7-108F7ED25D4E@umbi.umd.edu> Dear All, We are wondering about what kinds of red fluorescent reporters are good for making transgenic zebrafish. Our gene’s strongest expression occurs around 24hpf by in situ results. But when we used DsRed as the reporter we could not see any fluorescence until after 44-48hpf in our transgenic embryos. The fluorescence was pretty obvious at 24hpf if transient expression was checked by plasmid injection. So we guess the late detection of DsRed in these transgenic embryos is not because of the promoter we used is not complete or something. We thought this is might because of DsRed’s long maturation half time. We are thinking about using monomeric mCherry but worry about its low brightness compared to DsRed. If you have any experiences of making RFP transgenic fish please share them with us! Thanks in advance for your testimonies on this. Wei -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20091102/27628f88/attachment.html From Evert-Jan.van.den.Brandhof from rivm.nl Tue Nov 3 01:48:44 2009 From: Evert-Jan.van.den.Brandhof from rivm.nl (Evert-Jan van den Brandhof) Date: Tue Nov 3 12:29:39 2009 Subject: [Zbrafish] zebrafish gill disease? Message-ID: Skipped content of type multipart/alternative-------------- next part -------------- A non-text attachment was scrubbed... Name: kieuw effect.jpg Type: image/jpeg Size: 221001 bytes Desc: not available Url : http://www.bio.net/bionet/mm/zbrafish/attachments/20091103/aa9879ca/kieuweffect-0001.jpg From rburdine from Princeton.EDU Tue Nov 3 12:59:19 2009 From: rburdine from Princeton.EDU (Burdine, Rebecca D) Date: Tue Nov 3 13:03:53 2009 Subject: [Zbrafish] zebrafish gill disease? In-Reply-To: References: Message-ID: <790DAC09623D0A47ACDB126E3C52D8B63F35A2@MBCLUSTER.pu.win.princeton.edu> Our conductivity is routinely between 500-700 and we don't have an issue with this. Hope this helps, 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) 258-6730 Email: rburdine@princeton.edu Admin Assistant: Anna Schmedel (609) 258-5028 From: zbrafish-bounces@oat.bio.indiana.edu [mailto:zbrafish-bounces@oat.bio.indiana.edu] On Behalf Of Evert-Jan van den Brandhof Sent: Tuesday, November 03, 2009 1:49 AM To: zbrafish@magpie.bio.indiana.edu Subject: [Zbrafish] zebrafish gill disease? Hello, Is anyone familiar with (what looks as) fast growing epthelium on upper gill covers? We do'nt have any problem with egg production but it may cause some stress on breathing capacity? see picture: could anyone reconfirm this could be caused by conductivity? system: Zebtec (http://www.tecniplast.it/_assets/products/CAT012_ING_Internet.PDF) pH= 7.5 Conductivity= 500 ?S Temperature= 27,5 ?C Conductivity and pH are automatically adjusted with Seesalt and Sodiumbicarbonate. Thanks in advance, Evert-Jan ?????????????????????????????????????????????????? Evert-Jan van den Brandhof, BSc National Institute for Public Health and Environment (RIVM) Laboratory for Ecological Risk Assessment (LER) P.O. box 1, 3720 BA Bilthoven, The Netherlands Tel: (+31)30-2743544 http://www.rivm.nl/milieuportaal/ ?????????????????????????????????????????????????? Disclaimer RIVM -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20091103/a344812e/attachment.html From bobbi from aquaneering.com Tue Nov 3 20:46:52 2009 From: bobbi from aquaneering.com (Bobbi Baur) Date: Wed Nov 4 11:38:11 2009 Subject: [Zbrafish] Zebrafish Husbandry Workshop at WAS 2010 Message-ID: <0D939144273ADA4D8B38EC00A764E3190122D782@BE05.exg4.exghost.com> Skipped content of type multipart/alternative-------------- next part -------------- A non-text attachment was scrubbed... Name: not available Type: image/jpeg Size: 5650 bytes Desc: att513e5.jpg Url : http://www.bio.net/bionet/mm/zbrafish/attachments/20091103/95bb931c/attachment-0003.jpeg -------------- next part -------------- A non-text attachment was scrubbed... Name: not available Type: image/jpeg Size: 5425 bytes Desc: att513f6.jpg Url : http://www.bio.net/bionet/mm/zbrafish/attachments/20091103/95bb931c/attachment-0004.jpeg -------------- next part -------------- A non-text attachment was scrubbed... Name: not available Type: image/jpeg Size: 3092 bytes Desc: att513f7.jpg Url : http://www.bio.net/bionet/mm/zbrafish/attachments/20091103/95bb931c/attachment-0005.jpeg From gottalovelife from gmail.com Wed Nov 4 13:08:53 2009 From: gottalovelife from gmail.com (Kim) Date: Wed Nov 4 13:24:15 2009 Subject: [Zbrafish] Trizol extraction - zebrafish embryos Message-ID: <550ecde7-bb97-48e7-a327-3ae728dda614@13g2000prl.googlegroups.com> Has anyone encountered low 260/230 readings (<0.2) using the Trizol RNA extraction. We're getting okay yields and great purity using 5 embryos (24hpf-72hpf)/sample, but are having problems with what we assume is contamination. We have already tried a number of things: bought all our chemicals new, repeat chloroform extraction 2 times, do EtOH cleaning step 3 times. We homogenize manually with pestles. Thanks for any advice. From chi-bin.chien from neuro.utah.edu Wed Nov 4 13:51:12 2009 From: chi-bin.chien from neuro.utah.edu (Chi-Bin Chien) Date: Wed Nov 4 13:54:50 2009 Subject: [Zbrafish] Red fluorescent reporters for transgenic fish In-Reply-To: <4C727143-31AF-4701-9DC7-108F7ED25D4E@umbi.umd.edu> References: <4C727143-31AF-4701-9DC7-108F7ED25D4E@umbi.umd.edu> Message-ID: Dear Wei, Very likely your problem is due to the slow maturation of DsRed. mCherry should be better and is fairly bright. We have also had good success with TagRFP, which is extremely bright (available commercially from Evrogen). Another possibility is tdTomato, which is extremely bright. We have not used it ourselves but have heard good things from others. Don't know about maturation time. There is an easy experiment to test whether maturation time is the issue: do a DsRed in situ on your transgenics, and see if the timecourse mimics the native gene. best wishes, Chi-Bin Chien On Nov 2, 2009, at 5:19 PM, Wei Xia wrote: > Dear All, > We are wondering about what kinds of red fluorescent reporters are > good for making transgenic zebrafish. > Our gene’s strongest expression occurs around 24hpf by in situ > results. But when we used DsRed as the reporter we could not see any > fluorescence until after 44-48hpf in our transgenic embryos. The > fluorescence was pretty obvious at 24hpf if transient expression was > checked by plasmid injection. So we guess the late detection of > DsRed in these transgenic embryos is not because of the promoter we > used is not complete or something. We thought this is might because > of DsRed’s long maturation half time. We are thinking about using > monomeric mCherry but worry about its low brightness compared to > DsRed. > If you have any experiences of making RFP transgenic fish please > share them with us! > Thanks in advance for your testimonies on this. > Wei > > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20091104/ecc444bf/attachment.html From mikepfrank from gmail.com Tue Nov 17 14:24:23 2009 From: mikepfrank from gmail.com (Mike Frank) Date: Tue Nov 17 14:26:13 2009 Subject: [Zbrafish] In situ hybridization services for hire? Message-ID: <018c65d4-63ae-4078-ace8-b2bb19a7a6d4@z41g2000yqz.googlegroups.com> Hi, my lab just recently started experimenting with zebrafish as a model system for dermatologic diseases and I was wondering if anyone knew of any services/companies that do RNA in situ hybridization? If not, does anyone sell pre-made reagents? We're in a bit of a time crunch and while we already have a probe, we do not have the reagents needed and it just makes more sense to commission someone who has success with this procedure rather than us starting from scratch. From ilovequestions99 from gmail.com Tue Nov 17 16:44:31 2009 From: ilovequestions99 from gmail.com (Journey) Date: Tue Nov 17 18:11:12 2009 Subject: [Zbrafish] About embryo floating problem Message-ID: Sorry for the bothering! After the overnight hybridization when the embryos are transferred from Hyb to 2*SSC the embryos just start floating on the buffer. I tried to drop some buffer using pipets to immerse them into the water but when you changed the buffer the embryos are just floating again. Some of these embryos was not treated with proteinase K but they have the same problem. So I think it is not because of over digestion. Does anybody have the same problem before? How could you avoid this from happening? Thanks a lot for your help! Journey. -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20091117/4974f050/attachment.html From padnos from yahoo.com Wed Nov 18 09:12:32 2009 From: padnos from yahoo.com (padnos) Date: Wed Nov 18 12:13:01 2009 Subject: [Zbrafish] cd41-gfp and rag2-gfp Message-ID: I am looking for vendors and care tips for these lines From chubbyjayu from gmail.com Sun Nov 22 23:13:40 2009 From: chubbyjayu from gmail.com (chubbyjayu) Date: Mon Nov 23 12:23:57 2009 Subject: [Zbrafish] Alkaline Phosphatase staining of endothelial cells Message-ID: Hi there! My lab has been trying to target the alkaline phosphatase staining for the subintestinal basket. The protocol we follow is from Zebrafish: Nusslein-Volhard and Dahm.My problem is that although the basket gets stained, there's a lot of overstaining on the yolk, which does not allow for proper imaging. I have tried adjusting the amount of NBT/ BCIP I add, but to no avail. Could anyone tell me why the overstaining occurs, and how to prevent it? Thanks. From marina.raya from gmail.com Wed Nov 25 04:27:23 2009 From: marina.raya from gmail.com (Marina Raya) Date: Wed Nov 25 11:22:52 2009 Subject: [Zbrafish] plastic molds for microinjecting Zebrafish embryos Message-ID: Hi all I would like to get some plastic molds for microinjecting Zebrafish embryos (as the ones described in the ZF book, chapter 5, cellular methods, A device to Hold Zebrafish Embryos during microinjection), but I can not find any provider. Do you know where can I order or get them? From Caitlin.Stewart_swift from tufts.edu Wed Nov 25 12:21:02 2009 From: Caitlin.Stewart_swift from tufts.edu (Caitlin Stewart-Swift) Date: Wed Nov 25 12:56:44 2009 Subject: [Zbrafish] plastic molds for microinjecting Zebrafish embryos In-Reply-To: References: Message-ID: <4B0D677E.2010509@tufts.edu> This company makes the molds that you are interested in, our lab has purchased various models from them and they work very well: http://www.adaptivesciencetools.com/ -Caitlin Stewart-Swift Marina Raya wrote: > Hi all > I would like to get some plastic molds for microinjecting Zebrafish > embryos (as the ones described in the ZF book, chapter 5, cellular > methods, A device to Hold Zebrafish Embryos during microinjection), > but I can not find any provider. > Do you know where can I order or get them? > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish -- Caitlin Stewart-Swift Senior Research Technician/Zebrafish Facility Supervisor Department of Craniofacial and Molecular Genetics Tel(617)636-4011 Caitlin.Stewart_swift@tufts.edu