From gottalovelife from gmail.com Tue Apr 1 14:02:42 2008 From: gottalovelife from gmail.com (123) Date: Tue Apr 1 14:06:54 2008 Subject: [Zbrafish] Autofluorescence Message-ID: <7cf17242-ccbe-4cd2-a13c-2f94aaee9345@u10g2000prn.googlegroups.com> I'm having issues with autofluorescence interfering with my fluorescent and confocal imaging. Does anyone know at which wavelengths the fish will autofluoresce the least? I would like to be able to do my imaging on 4%PFA fixed fish, but I can do it live as well. I've already tried sodium borohydride but this does not quench the autofluorescence adequately. From jimdowlingmdphd from mac.com Thu Apr 3 13:30:21 2008 From: jimdowlingmdphd from mac.com (Jim D.) Date: Thu Apr 3 16:59:16 2008 Subject: [Zbrafish] dying embryos Message-ID: <4aa24b3c-7db1-4d6e-a245-b14bc0aa059d@m36g2000hse.googlegroups.com> Hello. We have recently had a problem. In our last several injections of either RNA or cDNA, every embryo has been dying. They look OK at 8 hpf but are dead by 24 hpf. At least one of the constructs has been tried previous and is not lethal by itself. One thought we had is that we are using a batch of Qiagen RNase free water for the dilutions. Otherwise, we are looking for thoughts/ suggestions. Has this all of sudden happened to anyone else? Thanks. From cheuk.chung from gmail.com Thu Apr 3 20:58:34 2008 From: cheuk.chung from gmail.com (Tony Cheuk-Chung Ho) Date: Fri Apr 4 12:29:14 2008 Subject: [Zbrafish] Re: dying embryos References: Message-ID: <9463b800-7d4d-4e49-ad07-da65d4599563@s19g2000prg.googlegroups.com> Jim, I have had experience injecting RNA into zebrafish embryos, and I think the water is a legit suspect. I would also look into the age of the fish used in producing the embryos and the pore size of the injection electrodes. In my experiments, I have noted that adult fish, that had been spawning (once a week) for nine months to a year or so, produced clutches that just die at gastrulation. This observation started out with at least half the clutch dying, and then rapidly deteriorated to ~70% of the clutch dying without any manipulation. Anther blunter in my injection experiments is the pore size on injection electrode, I used to have a batch of pretty big pored needle(~20um), they just trashed the injected embryos. I now use electrode with a pore size of about 7um and they work beautifully. Hope it helps. Good luck with the fish. Tony On Apr 4, 2:30?am, "Jim D." wrote: > Hello. ?We have recently had a problem. ?In our last several > injections of either RNA or cDNA, every embryo has been dying. ?They > look OK at 8 hpf but are dead by 24 hpf. ?At least one of the > constructs has been tried previous and is not lethal by itself. ?One > thought we had is that we are using a batch of Qiagen RNase free water > for the dilutions. ?Otherwise, we are looking for thoughts/ > suggestions. ?Has this all of sudden happened to anyone else? > Thanks. From chi-bin.chien from neuro.utah.edu Fri Apr 4 08:26:54 2008 From: chi-bin.chien from neuro.utah.edu (Chi-Bin Chien) Date: Fri Apr 4 12:29:43 2008 Subject: [Zbrafish] abstract deadline: 2008 international meeting Message-ID: 8th International Meeting on Zebrafish Development and Genetics June 25-29, 2008 - Madison, WI, USA To all of our zebrafish and medaka colleagues, A gentle reminder that the abstract submission deadline for the 2008 International Zebrafish meeting is coming up on Monday, April 14 (lodging deadline, May 19). You can find the meeting website here: http://www.union.wisc.edu/zebrafish/index.html In addition to keynote lectures by Chuck Kimmel and Allan Spradling, plenary and concurrent sessions, and poster sessions, this year we have added a new workshop format, whose topics were suggested by the community. These 10 workshops will generally feature a mixture of invited talks and talks selected from submitted abstracts, with significant time allotted for discussion. We think the workshop topics are very exciting and will be of interest to many in the community. Descriptions of the workshops can be found here: http://www.union.wisc.edu/zebrafish/workshops.html We are looking forward to seeing you and hearing about some great science in Madison. 2008 Organizing Committee: Sharon Amacher, UC Berkeley, USA Laure Bally-Cuif, GSF-Research Center for Environment and Health, Germany Chi-Bin Chien, University of Utah, USA Matthias Hammerschmidt, MPI Immunobiology, Germany Koichi Kawakami, National Institute of Genetics, Japan Brant Weinstein, NICHD/NIH, USA PS--Attached please find a poster announcing the meeting. Feel free to print it out and hang it up in your labs! -------------- next part -------------- Skipped content of type multipart/appledouble From stefan.weigt from merck.de Wed Apr 9 05:53:37 2008 From: stefan.weigt from merck.de (stefan1306) Date: Wed Apr 9 09:14:29 2008 Subject: [Zbrafish] egg production Message-ID: <000ef767-4760-4a70-89db-c929e323d598@d45g2000hsc.googlegroups.com> Hi everyone, I have got a problem with my zebrafish: the day before I want to collect eggs I put the egg traps in the aquarium (about 4 o?clock p. m.) and usually the animals spawn when the light is turned on the next day, but since several days the fish mainly spawn in the evening of the same day I put the traps in. And the next day when I collect the eggs they are to old for my experiments. I have got no idea what is the reason, perhaps someone can guess. Many thanks in advance! From hadie_k1984 from yahoo.com Wed Apr 9 15:42:43 2008 From: hadie_k1984 from yahoo.com (Hadie Khodabakhsh) Date: Thu Apr 10 11:38:56 2008 Subject: [Zbrafish] UCSF Mission Bay - Guo Lab Message-ID: <469021.20978.qm@web35505.mail.mud.yahoo.com> Good afternoon, I was wondering if you had any suggestions, or methods that will get zebrafish to mate more. I have already placed my zebrafish in the best environmental conditions, but they just don't seem to mate. So I was wondering if there were any hormonal treatment methods that can be used to influence the zebrafish to mate more? Thank you, Hadie Khodabakhsh (In case you are wondering, I am a research associate at the zebrafish lab of Dr. Guo at UCSF Mission Bay) __________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080409/07468137/attachment.html From meiw from ccri.net Wed Apr 9 09:36:38 2008 From: meiw from ccri.net (Wenyan Mei) Date: Thu Apr 10 11:39:28 2008 Subject: [Zbrafish] Breeding zebrafish Message-ID: Dear Jen, I am an investigator at the Ohio State University. We are having big problem with our fish breeding. It has been two and half years since my fish facility was built by Aquatic Habitats. The water condition is good in our fish facility: pH around 7.3-7.6, Conductivity around 250-300, and Temperature is a little higher (29C). We feed brine shrimp twice daily for the fish. Females look great with big belly. But they simply don't lay eggs for us. This really bother us because our project progress is delayed due to breeding problem. I am wondering if you can give me some suggestions to make fish breed better. Thanks a lot. wenyan ----------------------------------------- Confidentiality Notice: The following mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. The recipient is responsible to maintain the confidentiality of this information and to use the information only for authorized purposes. If you are not the intended recipient (or authorized to receive information for the intended recipient), you are hereby notified that any review, use, disclosure, distribution, copying, printing, or action taken in reliance on the contents of this e-mail is strictly prohibited. If you have received this communication in error, please notify us immediately by reply e-mail and destroy all copies of the original message. Thank you. From Christian.Lawrence from childrens.harvard.edu Thu Apr 10 11:57:35 2008 From: Christian.Lawrence from childrens.harvard.edu (Lawrence, Christian) Date: Thu Apr 10 12:05:36 2008 Subject: [Zbrafish] UCSF Mission Bay - Guo Lab In-Reply-To: <469021.20978.qm@web35505.mail.mud.yahoo.com> Message-ID: <1D117326B8F30943ACD9A47E1BEE29419A7EB6@CHEXV2.CHBOSTON.ORG> Hadie, No need for hormone treatments! There is much, much more to zebrafish reproduction than good water quality and putting a male and female together (as many would you have believe). Getting these fish to spawn isn't rocket science, but it does take careful consideration of a few key biological and behavioral attributes of the animal to be able to work through the occasional lulls in production. I would first check out the archives of this list as your question is probably to most common one posted to it. Many of the responses contain some good anecdotal information that will be helpful to you. If you have further questions, get in touch with me directly and I will be glad to assist you. Christian Lawrence Children's Hospital Boston Enders/ ARCH/Aquatics 320 Longwood Avenue Boston, MA 02115 Tel: 617.919.2738 christian.lawrence@childrens.harvard.edu ________________________________ From: zbrafish-bounces@oat.bio.indiana.edu [mailto:zbrafish-bounces@oat.bio.indiana.edu] On Behalf Of Hadie Khodabakhsh Sent: Wednesday, April 09, 2008 4:43 PM To: zbrafish@magpie.bio.indiana.edu Subject: [Zbrafish] UCSF Mission Bay - Guo Lab Good afternoon, I was wondering if you had any suggestions, or methods that will get zebrafish to mate more. I have already placed my zebrafish in the best environmental conditions, but they just don't seem to mate. So I was wondering if there were any hormonal treatment methods that can be used to influence the zebrafish to mate more? Thank you, Hadie Khodabakhsh (In case you are wondering, I am a research associate at the zebrafish lab of Dr. Guo at UCSF Mission Bay) __________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080410/db1d5f53/attachment.html From zfinadmn from zfin.org Thu Apr 10 12:53:56 2008 From: zfinadmn from zfin.org (zfinadmn@zfin.org) Date: Thu Apr 10 12:55:24 2008 Subject: [Zbrafish] Two funding opportunities at NIH Message-ID: NIH and the Trans-NIH Coordinating Committee have published two new Program Announcements with special Receipt dates (PARs) soliciting R01 applications for research using zebrafish. Please note that not all NIH Institutes and Centers are participating in both of these solicitations; therefore, only applications relevant to the missions of participating Institutes and Centers will be considered responsive to the announcements. These two new PAR's can be found in the NIH Guide at the following urls: http://grants.nih.gov/grants/guide/pa-files/PAR-08-138.html Genetic Screens to Enhance Zebrafish Research (R01) http://grants.nih.gov/grants/guide/pa-files/PAR-08-139.html Enhancing Zebrafish Research with Research Tools and Techniques (R01) From claudia_hohn from hotmail.com Fri Apr 11 08:30:44 2008 From: claudia_hohn from hotmail.com (Claudia) Date: Fri Apr 11 12:41:36 2008 Subject: [Zbrafish] Re: UCSF Mission Bay - Guo Lab References: Message-ID: <1211894b-5e1f-4011-b906-9c9b0f5bcc7c@8g2000hse.googlegroups.com> On Apr 9, 3:42 pm, Hadie Khodabakhsh wrote: > Good afternoon, > > I was wondering if you had any suggestions, or methods that will get zebrafish to mate more. I have already placed my zebrafish in the best environmental conditions, but they just don't seem to mate. So I was wondering if there were any hormonal treatment methods that can be used to influence the zebrafish to mate more? > > Thank you, > > Hadie Khodabakhsh > > (In case you are wondering, I am a research associate at the zebrafish lab of Dr. Guo at UCSF Mission Bay) > > __________________________________________________ > Do You Yahoo!? > Tired of spam? Yahoo! Mail has the best spam protection aroundhttp://mail.yahoo.com Hi Hadie, try keeping your males and females separate. Do not overfeed -if fish are too fat they do not reproduce either. We keep all our fish on a flow through system (to minimize contamination) which helps with elimination of pheromones too. Make sure your fish are not too old. We spawn fish that are 3 month to 1.5 years. Of course light cycle... is also important but you probably doing all that already. If you need to built a cheap flow though system to hold your broodstock check out my following article: * Hohn, C.M., Petrie-Hanson, L. (2007): Low cost aquatic lab animal holding system. Zebrafish 4(2):117-122. Hope this helps, Claudia From mwinandy from ulg.ac.be Fri Apr 11 01:23:58 2008 From: mwinandy from ulg.ac.be (Marie Winandy) Date: Fri Apr 11 12:41:58 2008 Subject: [Zbrafish] Re: Zbrafish Digest, Vol 35, Issue 5 In-Reply-To: <200804101704.m3AH4OT25047@net.bio.net> References: <200804101704.m3AH4OT25047@net.bio.net> Message-ID: <200804110823580125.000E4EF6@smtp.ulg.ac.be> Hi all, Some diets with high protein content work well. We experienced them on mutants strains that were not laying very well and the mating rate improved within 4 weeks. Remember also that"old" fish don't lay well (> 14 months). You can also keeep males and females separated during one week to allow eggs accumulation by females. When you 'll do the next mating, you should get more eggs. Also, in my experience, some strains are (very) good layers and other not. Good luck! Marie Winandy, PhD Université de Liège GIGA B34 - Zebrafish Platform Avenue de l'Hôpital 1 B-4000 Liège - Sart Tilman Belgique Tél: +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 *********** REPLY SEPARATOR *********** On 10/04/2008 at 12:04 zbrafish-request@oat.bio.indiana.edu 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. UCSF Mission Bay - Guo Lab (Hadie Khodabakhsh) > 2. Breeding zebrafish (Wenyan Mei) > > >---------------------------------------------------------------------- > >Message: 1 >Date: Wed, 9 Apr 2008 13:42:43 -0700 (PDT) >From: Hadie Khodabakhsh >Subject: [Zbrafish] UCSF Mission Bay - Guo Lab >To: zbrafish@magpie.bio.indiana.edu >Message-ID: <469021.20978.qm@web35505.mail.mud.yahoo.com> >Content-Type: text/plain; charset="iso-8859-1" > >Good afternoon, > >I was wondering if you had any suggestions, or methods that will get >zebrafish to mate more. I have already placed my zebrafish in the best >environmental conditions, but they just don't seem to mate. So I was >wondering if there were any hormonal treatment methods that can be used to >influence the zebrafish to mate more? > >Thank you, > >Hadie Khodabakhsh > >(In case you are wondering, I am a research associate at the zebrafish lab >of Dr. Guo at UCSF Mission Bay) > > __________________________________________________ >Do You Yahoo!? >Tired of spam? Yahoo! Mail has the best spam protection around >http://mail.yahoo.com >-------------- next part -------------- >An HTML attachment was scrubbed... >URL: >http://www.bio.net/bionet/mm/zbrafish/attachments/20080409/07468137/attachment-0001.html > >------------------------------ > >Message: 2 >Date: Wed, 09 Apr 2008 10:36:38 -0400 >From: Wenyan Mei >Subject: [Zbrafish] Breeding zebrafish >To: >Message-ID: >Content-Type: text/plain; charset="US-ASCII" > >Dear Jen, > >I am an investigator at the Ohio State University. We are having big >problem >with our fish breeding. It has been two and half years since my fish >facility was built by Aquatic Habitats. The water condition is good in our >fish facility: pH around 7.3-7.6, Conductivity around 250-300, and >Temperature is a little higher (29C). We feed brine shrimp twice daily for >the fish. Females look great with big belly. But they simply don't lay eggs >for us. This really bother us because our project progress is delayed due >to >breeding problem. I am wondering if you can give me some suggestions to >make >fish breed better. Thanks a lot. >wenyan > >----------------------------------------- Confidentiality Notice: >The following mail message, including any attachments, is for the >sole use of the intended recipient(s) and may contain confidential >and privileged information. The recipient is responsible to >maintain the confidentiality of this information and to use the >information only for authorized purposes. If you are not the >intended recipient (or authorized to receive information for the >intended recipient), you are hereby notified that any review, use, >disclosure, distribution, copying, printing, or action taken in >reliance on the contents of this e-mail is strictly prohibited. If >you have received this communication in error, please notify us >immediately by reply e-mail and destroy all copies of the original >message. Thank you. > > > >------------------------------ > >_______________________________________________ >Zbrafish mailing list >Zbrafish@net.bio.net >http://www.bio.net/biomail/listinfo/zbrafish > >End of Zbrafish Digest, Vol 35, Issue 5 >*************************************** From tgenade from gmail.com Fri Apr 11 03:10:15 2008 From: tgenade from gmail.com (Tyrone) Date: Fri Apr 11 12:42:21 2008 Subject: [Zbrafish] Re: Breeding zebrafish References: Message-ID: Hello Wenyan, > ...We are having big problem with our fish breeding... > fish facility: pH around 7.3-7.6, Conductivity around 250-300, and > Temperature is a little higher (29C)... But they simply don't lay eggs Your temperature is too high for breeding. This is how I used to spawn my zebra danios in large numbers (to feed to other more carnivorous fish fry). 1. seperate the males and females and condition them for 3 days. 2. prepeare a tank with either marbels on the bottom or a fine plastic mesh that can seperate the eggs from the fish (which will not hesitate to eat the eggs). You want about 5 cm of water above the mess or marbels. A fine mesh with 5 mm pores works best in my opinion. 1 cm below the mesh is fine. 3. in the evening bring the males and females together (2 males per female) and then do a 50% water change with cool water. A drop in water temperature stimulates spawning. 10 deg C drop is perfectly fine. The water temperature must not exceed 24 deg C for breeding. The next morning the fish WILL spawn. 4. remove the fish after spawning and you can take out the plastic mesh too. 5. keep doing 50% water changes on the spawning tank or pour the eggs off into an incubator that will ensure that they are kept well oxygenated and in clean water. Some people suggest the use of anti- fungal or anti-bacterial agents to protect the eggs but most of these are DNA intercalating agents and I have always gotten away without using them. Clean well oxygenated water seems to be all you need to ensure high egg survival. For the record, I do not work with zebrafish in any professional capacity now or ever but for almost 20 years I have kept and spawned them in pretty good numbers. Best of luck Tyrone From wilson.clements from gmail.com Sat Apr 12 12:13:50 2008 From: wilson.clements from gmail.com (Wilson Clements) Date: Mon Apr 14 11:31:25 2008 Subject: [Zbrafish] Re: Breeding Problems References: <5600B8F5-7FB1-43AF-8954-751DF6291890@ucsd.edu> Message-ID: <93DFE36D-8690-4382-A97C-E81CF62FFFB1@gmail.com> > Dear Drs. Khodabakhsh and Mei, > > With respect to your breeding problems, we went through similar low > fecundity here and tried numerous things. We found that the thing > that made the greatest difference was feeding. Here was our > situation: > > Fish were optimal age (6 mos. to 1.2 years old). Water quality was > good, with pH running around 7.1 to 7.2 and conductivity at around > 400 to 600 uS. We tested for nitrites, nitrates, and ammonia and > found our levels to be reasonable. Still we were having fertility > issues. > > We changed our feeding schedule to have two brine shrimp feedings > per day with 1 liter of brine shrimp per rack, hatched at 6g of > cysts per liter. On week days, individuals feed their racks with > an evening supplemental feeding of "Scientific Hatcheries 3 Pigment > Diet/ Fish Food for Zebrafish/Danio Rerio" Product # SFF1 from > Aquaneering, 7960 Stromesa Ct., San Diego, CA, 92126, USA > 858-578-2028. I generally feed my fish enough food that they can > clear the top surface of the water in approximately 2 minutes. > > Since we have adopted this feeding routine, 60-90% of my crosses > are productive. > > Here are additional things we tried that seem to make some limited > difference: > > 1) Change water in the morning prior to breeding. > 2) Shield fish from room activity (but not light!). > 3) Marbles and/or fake seaweed in the breeding chamber. > 4) Angle breeding chambers to create a "shallow" end. > > I also read that providing new water with slightly lower > conductivity (~200-300 uS) in the morning of breeding helped. This > was not a feasible option for us, so we have never tried it, and I > don't know if it works. > > Hope this helps. > > Best, > Wilson > ---------------------------------------------------------------------- > ---------------------- > Wilson Clements, Ph.D. > > wclements@ucsd.edu > > Dept. of Biology > Section of Cell and Developmental Biology > University of California at San Diego > 9500 Gilman Dr. > Natural Sciences Building 6105 > La Jolla, CA 92093-0380 > > TEL (858) 534-6955 > LAB (858) 822-4658 > FAX (858) 822-5740 > > > Begin forwarded message: >> Today's Topics: >> >> 1. UCSF Mission Bay - Guo Lab (Hadie Khodabakhsh) >> 2. Breeding zebrafish (Wenyan Mei) >> >> >> --------------------------------------------------------------------- >> - >> >> Message: 1 >> Date: Wed, 9 Apr 2008 13:42:43 -0700 (PDT) >> From: Hadie Khodabakhsh >> Subject: [Zbrafish] UCSF Mission Bay - Guo Lab >> To: zbrafish@magpie.bio.indiana.edu >> Message-ID: <469021.20978.qm@web35505.mail.mud.yahoo.com> >> Content-Type: text/plain; charset="iso-8859-1" >> >> Good afternoon, >> >> I was wondering if you had any suggestions, or methods that will >> get zebrafish to mate more. I have already placed my zebrafish in >> the best environmental conditions, but they just don't seem to >> mate. So I was wondering if there were any hormonal treatment >> methods that can be used to influence the zebrafish to mate more? >> >> Thank you, >> >> Hadie Khodabakhsh >> >> (In case you are wondering, I am a research associate at the >> zebrafish lab of Dr. Guo at UCSF Mission Bay) >> >> __________________________________________________ >> Do You Yahoo!? >> Tired of spam? Yahoo! Mail has the best spam protection around >> http://mail.yahoo.com >> -------------- next part -------------- >> An HTML attachment was scrubbed... >> URL: http://www.bio.net/bionet/mm/zbrafish/attachments/ >> 20080409/07468137/attachment-0001.html >> >> ------------------------------ >> >> Message: 2 >> Date: Wed, 09 Apr 2008 10:36:38 -0400 >> From: Wenyan Mei >> Subject: [Zbrafish] Breeding zebrafish >> To: >> Message-ID: >> Content-Type: text/plain; charset="US-ASCII" >> >> Dear Jen, >> >> I am an investigator at the Ohio State University. We are having >> big problem >> with our fish breeding. It has been two and half years since my fish >> facility was built by Aquatic Habitats. The water condition is >> good in our >> fish facility: pH around 7.3-7.6, Conductivity around 250-300, and >> Temperature is a little higher (29C). We feed brine shrimp twice >> daily for >> the fish. Females look great with big belly. But they simply don't >> lay eggs >> for us. This really bother us because our project progress is >> delayed due to >> breeding problem. I am wondering if you can give me some >> suggestions to make >> fish breed better. Thanks a lot. >> wenyan > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080412/bd8a3f16/attachment.html From mahaffy from dordt.edu Tue Apr 15 11:02:07 2008 From: mahaffy from dordt.edu (James) Date: Tue Apr 15 11:14:35 2008 Subject: [Zbrafish] embryo decrease Message-ID: Folks, We had a student who was doing a project using Zebra fish embryo's this summer. At first he got pretty good embryo yields but as this semester progressed he got less and less until we got almost none at the end of this semester. The fish were fed well and we should have had fairly good water so I am not sure what was the problem. Do the fish just slow down over time and you have to start with fresh fish or fish that are kept under different light conditions? We had 14 light and 10 dark and put in a trap when we wanted to collect embryo's and we had about 10 adults for each of two tanks. I am a little worried because we have a student who will be doing this again in the Fall and we would like to be sure that we get plenty of embryo's. Any suggestions on how to keep the numbers of embryo's high? You can respond directly back to me at mahaffy@dordt.edu or to the list. He used the following setup: The test organism for my project was Danio rerio. The fish were acquired from the Dordt College biology department who originally obtained them from The Dog House Etc in Sheldon. The fish were housed in two separate tanks to help increase embryo collection numbers. Each tank was approximately 10 gallons and was maintained at around 28.5 degrees Celsius for optimum embryo production. The fish were fed a rotation of fish flakes with brine shrimp or with blood worms. This diet encouraged more vigorous embryo production. It was better for the fish to be fed less food at more frequent intervals. The fish were maintained and bred according to accepted methods in The Zebrafish Book (Westerfield 2000). Frequent water cleaning (rotation) promoted embryo production. I changed over half of the water on a weekly basis (Westerfield 2000, Warolin 2002). From jocelyn.mcauley from gmail.com Tue Apr 15 13:30:26 2008 From: jocelyn.mcauley from gmail.com (Jocelyn) Date: Tue Apr 15 13:34:34 2008 Subject: [Zbrafish] Re: Breeding zebrafish References: Message-ID: On Apr 11, 1:10 am, Tyrone wrote: > Hello Wenyan, > > > ...We are having big problem with our fish breeding... > > fish facility: pH around 7.3-7.6, Conductivity around 250-300, and > > Temperature is a little higher (29C)... But they simply don't lay eggs > > Your temperature is too high for breeding. This is how I used to spawn > my zebra danios in large numbers (to feed to other more carnivorous > fish fry). For the record, I disagree. Our zebrafish breed fine at 29C. Try the simplest methods first. From rathmark from mail.nih.gov Tue Apr 15 13:31:57 2008 From: rathmark from mail.nih.gov (marcuswrath) Date: Tue Apr 15 13:35:21 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways Message-ID: I'm throwing this out to the Internet to see if anyone has any unusual solutions to this problem. I'm trying to come up with an alternative to the traditional foot baths and tacky-mats to be used at the entry points to a zebrafish room. I'm stuck with the following restrictions: 1) Foot baths are out of the question due to "investigator dislike" 2) The doors to the fish room open into the room and will have door sweeps flush with the floor (making it impossible to set something on the floor just inside the doors) 3) The exterior hallway is a shared hallway that will have lots of non- aquatics personnel. If anyone has any ideas other than shoe covers for reducing the likelihood of unwanted material being carried in on people's shoes, I'd love to hear them! From jbennett from bio.umass.edu Tue Apr 15 14:06:07 2008 From: jbennett from bio.umass.edu (Judy Bennett) Date: Tue Apr 15 14:06:09 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways In-Reply-To: References: Message-ID: <200804151903.m3FJ3Xtn003978@marlin.bio.umass.edu> Hi Marcus, We had an issue with "pond organisms" turning up in one of our 3 fish rooms. To prevent further contamination, or spreading, we implemented foot baths using a broad spectrum disinfectant (we have a lot of foot traffic in our halls). They're set just inside each doorway for folks to use on their way in and out. There is minimal maintenance involved and I believe it's got to be part of our bio-security protocol. I'm surprised that an investigator would rather opt for shoe covers? I'm interested to see what inventive solutions are out there, but if nothing practical comes up, I would have a discussion with the investigator as to why foot baths need to be part of the facility's bio-security. Good luck, Judy At 02:31 PM 4/15/08, marcuswrath wrote: >I'm throwing this out to the Internet to see if anyone has any unusual >solutions to this problem. I'm trying to come up with an alternative >to the traditional foot baths and tacky-mats to be used at the entry >points to a zebrafish room. I'm stuck with the following restrictions: > >1) Foot baths are out of the question due to "investigator dislike" >2) The doors to the fish room open into the room and will have door >sweeps flush with the floor (making it impossible to set something on >the floor just inside the doors) >3) The exterior hallway is a shared hallway that will have lots of non- >aquatics personnel. > >If anyone has any ideas other than shoe covers for reducing the >likelihood of unwanted material being carried in on people's shoes, >I'd love to hear them! > >_______________________________________________ >Zbrafish mailing list >Zbrafish@net.bio.net >http://www.bio.net/biomail/listinfo/zbrafish From dgw5079 from u.washington.edu Tue Apr 15 14:44:34 2008 From: dgw5079 from u.washington.edu (David G. White) Date: Tue Apr 15 14:46:17 2008 Subject: [Zbrafish] Re: Breeding zebrafish In-Reply-To: Message-ID: Make sure they are being fed a proper diet, we use small salmon feed and artemia (there should always be at least two types of feed). Your temperature should not be a problem, but I would adjust it down to 28c if you can. With a pH that can reach 7.6 and a slightly high temperature I would check your toxic ammonia and nitrite levels or if applicable remove some coral from the system you might have some hard water. In a normal functioning water recirculating system you should have to "fight" to keep the pH just above 7.0. Optimal breeding age is 6 months to 1.5 years. If you have had a normal operating system before where the fish breeding was good look to see what is different now. Water quality, feed, breeding techniques, age, health of the fish. In general, check your environment for what is different. David G White Research Coordinator H225 Zebrafish Lab University of Washington Department of Biological Structure HSB G514 Box 357420 1959 NE Pacific Street Seattle, WA 98195-7420 Tel. 206-685-7512 FAX 206-543-1524 On Tue, 15 Apr 2008, Jocelyn wrote: > On Apr 11, 1:10 am, Tyrone wrote: >> Hello Wenyan, >> >>> ...We are having big problem with our fish breeding... >>> fish facility: pH around 7.3-7.6, Conductivity around 250-300, and >>> Temperature is a little higher (29C)... But they simply don't lay eggs >> >> Your temperature is too high for breeding. This is how I used to spawn >> my zebra danios in large numbers (to feed to other more carnivorous >> fish fry). > > For the record, I disagree. Our zebrafish breed fine at 29C. > > Try the simplest methods first. > > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > From Christian.Lawrence from childrens.harvard.edu Tue Apr 15 15:10:18 2008 From: Christian.Lawrence from childrens.harvard.edu (Lawrence, Christian) Date: Tue Apr 15 15:16:32 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways Message-ID: <1D117326B8F30943ACD9A47E1BEE29419A7F0F@CHEXV2.CHBOSTON.ORG> Seems to me that the easiest solution would be dedicated shoes. Rubber clogs like anywears or crocs work well. You can set up an area to make the swap just outside or inside the room, utilizing a tacky mat to help cut dirt down at the entrance. ----- Original Message ----- From: zbrafish-bounces@oat.bio.indiana.edu To: bionet-organisms-zebrafish@moderators.isc.org Sent: Tue Apr 15 14:31:57 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways I'm throwing this out to the Internet to see if anyone has any unusual solutions to this problem. I'm trying to come up with an alternative to the traditional foot baths and tacky-mats to be used at the entry points to a zebrafish room. I'm stuck with the following restrictions: 1) Foot baths are out of the question due to "investigator dislike" 2) The doors to the fish room open into the room and will have door sweeps flush with the floor (making it impossible to set something on the floor just inside the doors) 3) The exterior hallway is a shared hallway that will have lots of non- aquatics personnel. If anyone has any ideas other than shoe covers for reducing the likelihood of unwanted material being carried in on people's shoes, I'd love to hear them! _______________________________________________ Zbrafish mailing list Zbrafish@net.bio.net http://www.bio.net/biomail/listinfo/zbrafish The information transmitted in this electronic communication is intended only for the person or entity to whom it is addressed and may contain confidential and/or privileged material. Any review, retransmission, dissemination or other use of or taking of any action in reliance upon this information by persons or entities other than the intended recipient is prohibited. If you received this information in error, please contact the Compliance HelpLine at 800-856-1983 and properly dispose of this information. From waldrons from u.washington.edu Tue Apr 15 10:41:40 2008 From: waldrons from u.washington.edu (Steven Waldron) Date: Tue Apr 15 17:27:36 2008 Subject: [Zbrafish] embryo decrease In-Reply-To: References: Message-ID: <4804CCB4.2010009@u.washington.edu> Hi James, You might want to make sure your fish are not too old too breed. After they are a year old or so their productivity can become capricious. Separating the sexes from on another can also increase yields. The fish could very well be breeding in their holding tanks before you ever set up them up to spawn. best, Steve Waldron University of Washington, Seattle James wrote: > Folks, > > We had a student who was doing a project using Zebra fish embryo's > this summer. > > At first he got pretty good embryo yields but as this semester > progressed he got less and less until we got almost none at the end of > this semester. The fish were fed well and we should have had fairly > good water so I am not sure what was the problem. Do the fish just > slow down over time and you have to start with fresh fish or fish that > are kept under different light conditions? > > We had 14 light and 10 dark and put in a trap when we wanted to > collect embryo's and we had about 10 adults for each of two tanks. > > I am a little worried because we have a student who will be doing this > again in the Fall and we would like to be sure that we get plenty of > embryo's. Any suggestions on how to keep the numbers of embryo's > high? > > You can respond directly back to me at mahaffy@dordt.edu or to the > list. > > He used the following setup: > > The test organism for my project was Danio rerio. The fish were > acquired from the Dordt College biology department who originally > obtained them from The Dog House Etc in Sheldon. The fish were housed > in two separate tanks to help increase embryo collection numbers. Each > tank was approximately 10 gallons and was maintained at around 28.5 > degrees Celsius for optimum embryo production. The fish were fed a > rotation of fish flakes with brine shrimp or with blood worms. This > diet encouraged more vigorous embryo production. It was better for the > fish to be fed less food at more frequent intervals. The fish were > maintained and bred according to accepted methods in The Zebrafish > Book (Westerfield 2000). Frequent water cleaning (rotation) promoted > embryo production. I changed over half of the water on a weekly basis > (Westerfield 2000, Warolin 2002). > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > From dykvar from yahoo.com Fri Apr 18 13:34:55 2008 From: dykvar from yahoo.com (dykvar@yahoo.com) Date: Fri Apr 18 13:36:39 2008 Subject: [Zbrafish] Sequence homology for protein expression - help! Message-ID: <9da02037-1cd7-4aee-a587-463a246279c4@e39g2000hsf.googlegroups.com> Hi, I have a general question regarding rescue and protein expression via construct in ZF. To do this one must first clone the protein mRNA sequence from cDNA, then insert into an expression vector (I’m using PCS2+). However, whenever I clone and then sequence I never get 100% homology when blasting on NCBI. Although I use high-fidelity (Roche) enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa different from what is in the database. Is this normal? Is it a result of polymorphism? Or is it a problem with my methodology? And what degree of homology is needed (assuming no gaps exist) for the protein being expressed properly? Your help is appreciated!! From austin from rowellbrokaw.com Mon Apr 21 11:22:04 2008 From: austin from rowellbrokaw.com (Austin Bailey) Date: Mon Apr 21 12:02:06 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways Message-ID: <28226342-0461-42B9-AA15-D5AB8E4606E0@rowellbrokaw.com> Your not alone in this search. Barrier strategies are definitely some of the most difficult aspects to retrofit for an existing facility. For this reason, something completely independent of the building infrastructure is usually recommended. Dedicated shoes fall into this category. You'd still need to confirm that you have space for a changing area. Because of the traffic outside the room, I'd recommend swapping shoes inside the room. This will keep the aquatic shoes as clean as possible. But any strategy needs to be viewed in the context of the overall containment and barrier strategies for the facility. Pathogen control is only as good as its weakest link. Although they may be warranted in certain cases, strategies like disinfectant shoe baths are a holdover from the rodent industry. Most installations I've seen allow users to often step over or around the mats. With proper floor and hand sanitization, shoe sterilization might not be as necessary. And if other barriers are not in place, foot sanitization may be a moot point. The most effective strategies for barrier to pathogens involve unidirectional workflow coupled with those primary barriers such as foot baths and hand sanitization. If there are multiple entrances to your facility this may be something to consider. If you have only a single entrance, then foot sanitization becomes much less effective due to the cross traffic in, out and past the single entrance - even with a walk-off mat in place. It may be much more effective in your case to focus on regular sanitization of the floor and other established methods of pathogen control. Austin Bailey Rowell Brokaw Architects, PC 1 East Broadway, Suite 300 Eugene, OR 97401 tel: 541.485.1003 www.rowellbrokaw.com From joanna.wilson from mcmaster.ca Sun Apr 20 07:07:58 2008 From: joanna.wilson from mcmaster.ca (Joanna Wilson) Date: Mon Apr 21 12:02:51 2008 Subject: [Zbrafish] Re:Sequence homology for protein expression - help! Message-ID: <5C44B3F5-4A6F-4F3C-8915-B5EB55F7E5AA@mcmaster.ca> Hi there, I am totally unsurprised that you are finding sequence differences with the gene you cloned and what is in NCBI. We see this in the genes we clone in the lab - one to 10 nucleotide sequence differences (our genes are 1500 bp) with aa changes. You can see this if you pull out sequence information from NCBI for which there are multiple entries - they will not be completely identical. Some of this is strain differences. It doesn't mean it won't be expressed. You need to be sure that the differences are real (sequence several clones so that you can show it isn't a PCR or sequencing error) and that it will translate in frame without stops. Joanna Wilson Re: Message: 1 Date: Fri, 18 Apr 2008 11:34:55 -0700 (PDT) From: dykvar@yahoo.com Subject: [Zbrafish] Sequence homology for protein expression - help! To: bionet-organisms-zebrafish@moderators.isc.org Message-ID: <9da02037-1cd7-4aee-a587-463a246279c4@e39g2000hsf.googlegroups.com> Content-Type: text/plain; charset=windows-1252 Hi, I have a general question regarding rescue and protein expression via construct in ZF. To do this one must first clone the protein mRNA sequence from cDNA, then insert into an expression vector (I?m using PCS2+). However, whenever I clone and then sequence I never get 100% homology when blasting on NCBI. Although I use high-fidelity (Roche) enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa different from what is in the database. Is this normal? Is it a result of polymorphism? Or is it a problem with my methodology? And what degree of homology is needed (assuming no gaps exist) for the protein being expressed properly? Your help is appreciated!! Dr. Joanna Wilson Assistant Professor Department of Biology McMaster University 1280 Main Street West Hamilton ON L8S 4K1 Tel: 905-525-9140 ext 20075 Fax: 905-522-6066 joanna.wilson@mcmaster.ca -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080420/b2672800/attachment.html From rburdine from Princeton.EDU Mon Apr 21 17:00:36 2008 From: rburdine from Princeton.EDU (Burdine, Rebecca D) Date: Mon Apr 21 17:16:12 2008 Subject: [Zbrafish] Suggestions for fish room entry-ways In-Reply-To: <28226342-0461-42B9-AA15-D5AB8E4606E0@rowellbrokaw.com> References: <28226342-0461-42B9-AA15-D5AB8E4606E0@rowellbrokaw.com> Message-ID: <04AF14C4EF3CF34682460FB1FDBA98C00F105E@MBCLUSTER.pu.win.princeton.edu> Hi everyone, We don't use foot baths or dedicated shoes for our facility. Our fish facility is in a larger hallway that contains other animal care rooms, but our rooms are separate. There are sticky mats when you enter the main hallway, but I am not sure these help with anything to be honest. Our policy is that if a fish hits the floor, it has to be euthanized. We ask everyone coming into the facility to wash their hands when the enter (soap without Triclosan) and when they leave. We also have Purell for anyone feeling the need. In general, our policy to help keep down contamination is that no new fish enter our system. They must all come from bleached eggs or they go into the Q-room. We don't take equipment in or out of the facility and definitely don't mix between the Qroom and the main facility. We have an incubator specifically for embryos from our Q-Room. Anything in that incubator cannot go back into the main facility. We also use commercial shrimp eggs and food for our dry mixes that is free of pathogens. Nothing from a pet store is fed to or added to our system. But to be honest, I think the main things we do that keep our facility disease free is keep everything as clean as possible (tanks, floors, and equipment) and make sure fish that are 2+ years old or fish that start to look unhealthy are removed from the facility promptly. It is the Logan's Run approach to fish health, but it has worked so far. (Knock on wood.) Many of the practices that make sense for a rodent facility either aren't useful or are down right dangerous in a fish facility. The IACUC here is very reasonable, but I had to write a long letter explaining why certain practices don't make sense for us. For example, they were asking why we don't wear gloves, gowns, masks and booties. I asked if their concern was for the animals or for the people working with the animals. They were very frank and told me they only care about the health and well-being of the animals. So I explained that my job is to care for both. Booties are a slip hazard and dangerous to my workers. Gowns and gloves can be accidently dragged into fish tanks or set up cages which can contaminate them. In the end, since we aren't concerned with passing things back and forth between the fish and the students, all the gowning does is make the students uncomfortable, hotter than they should be with the temperature, and can actually contaminate the fish tanks. Likewise we had to argue that we would prefer to see some rust as opposed to having paint aerosolize near our precious fish! You have to do what is the most comfortable for your system and setups, but we also need to help educate IACUCs around the world about what is necessary for fish, and what is not. Otherwise we may be paying technicians to entertain our fish and provide "stimulating environments" before you know it. Just my 2 cents worth. 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 From rathmark from mail.nih.gov Tue Apr 22 13:47:53 2008 From: rathmark from mail.nih.gov (marcuswrath) Date: Tue Apr 22 13:53:00 2008 Subject: [Zbrafish] Re: Suggestions for fish room entry-ways References: Message-ID: Thanks for your input, everyone (and to those who replied off-list). -Mark Mark A. Rath Aquatic Specialist IV Charles River/NIH-Contractor 14G/104A 14 Service Rd. South MSC 5590 Bethesda, MD 20892-5590 Cell phone: (410) 428-9851 Fax: (301) 480-1317 E-mail: rathmark@mail.nih.gov From dykvar from yahoo.com Wed Apr 23 10:52:17 2008 From: dykvar from yahoo.com (dykvar@yahoo.com) Date: Wed Apr 23 12:00:32 2008 Subject: [Zbrafish] Re: Sequence homology for protein expression - help! References: Message-ID: <3985ceeb-43e5-4ec6-8041-1715c6c8f01d@m44g2000hsc.googlegroups.com> On Apr 20, 8:07 am, Joanna Wilson wrote: > Hi there, > I am totally unsurprised that you are finding sequence differences   > with the gene you cloned and what is in NCBI.  We see this in the   > genes we clone in the lab - one to 10 nucleotide sequence differences   > (our genes are 1500 bp) with aa changes.  You can see this if you pull   > out sequence information from NCBI for which there are multiple   > entries - they will not be completely identical.  Some of this is   > strain differences.  It doesn't mean it won't be expressed.  You need   > to be sure that the differences are real (sequence several clones so   > that you can show it isn't a PCR or sequencing error) and that it will   > translate in frame without stops. > Joanna Wilson > > Re: > Message: 1 > Date: Fri, 18 Apr 2008 11:34:55 -0700 (PDT) > From: dyk...@yahoo.com > Subject: [Zbrafish] Sequence homology for protein expression - help! > To: bionet-organisms-zebraf...@net.bio.net > Message-ID: >         <9da02037-1cd7-4aee-a587-463a24627...@e39g2000hsf.googlegroups.com> > Content-Type: text/plain; charset=windows-1252 > > Hi, > I have a general question regarding rescue and protein expression via > construct in ZF. To do this one must first clone the protein mRNA > sequence from cDNA, then insert into an expression vector (I’m using > PCS2+). However, whenever I clone and then sequence I never get 100% > homology when blasting on NCBI. Although I use high-fidelity (Roche) > enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa > different from what is in the database. > Is this normal? Is it a result of polymorphism? Or is it a problem > with my methodology? And what degree of homology is needed (assuming > no gaps exist) for the protein being expressed properly? > Your help is appreciated!! > > Dr. Joanna Wilson > Assistant Professor > Department of Biology > McMaster University > > 1280 Main Street West > Hamilton ON > L8S 4K1 > > Tel: 905-525-9140 ext 20075 > Fax: 905-522-6066 > joanna.wil...@mcmaster.ca Hi Joanna, Thanks for the answer. The strange thing is that I do not get the same ‘errors’ when sequencing multiple clones. I can only think that this is due to the fact that the cDNA was made from multiple larvae…? Or that although I used HF the enzyme is causing errors. In any case, when I use blastx for protein sequence (part of the protein – 900bp) I get something like this: Score = 446 bits (1147), Expect = 1e-123 Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221 (0%) Frame = +2 (One alanine substituting a threonine) Is such a substitution a problem? Thanks very much E.A. From wclements from ucsd.edu Wed Apr 23 13:21:34 2008 From: wclements from ucsd.edu (Wilson Clements) Date: Wed Apr 23 13:24:22 2008 Subject: [Zbrafish] Re: Sequence homology for protein expression References: <200804231703.m3NH3QT06787@net.bio.net> Message-ID: <3352EC09-A924-4298-B291-31C860264DCA@ucsd.edu> Re: Sequence homology for protein expression Dear E.A., I would say that to be careful, you should consider different amino acid substitutions in different clones to be a problem. For the following reasons: 1) The fact that you are not getting the same errors clone-to-clone strongly suggests that you are getting PCR errors. High fidelity enzymes are not perfect. Are you running high numbers of amplification cycles (>30-32)? The less cycles, the less errors you should get, although you can still be unlucky and get errors at low numbers of cycles. You could try: using a variety of enzymes, I now prefer Phusion (from Finnzymes) and iProof (from Bio-Rad), they seem to give me the lowest number of errors. Making all your cDNA from a single clutch from a pairwise cross. Then you will know that you should get very few polymorphisms clone-to-clone. 2) Many amino acid substitutions don't matter, but without having the "wild-type" to compare functionality against, it will be hard to be sure yours doesn't. Especially a Threonine to Alanine substitution, since Thr is frequently a phosphorylated amino acid for protein regulation. If I start getting "polymorphisms" different from the expected amino acid sequence, I sequence quite a few more clones to make sure they all have the same substitutions. I don't worry so much about silent base substitutions (in the third position of the codon leading to no change in amino acid). In general, I don't think pairwise BLAST is a very good way to assess whether your clones are good. The BLAST algorithm tends to cut off ends or regions that are "not conserved" so you don't get a base-by- base comparison. We use the Sequencher software here. I'm not sure if you have access to it, but many universities and companies have purchased networked licenses that you can use on a client/server basis using downloaded software. I think it's the best thing going. You can make a "virtual" clone of your expected sequence (in the vector) and then do an alignment (make a contig) against the actual sequencing returned. Then you can look base-by-base at which bases match and which do not. In addition, you can consider the quality of the sequencing data. If your "polymorphisms" are towards the end, or very beginning, of a sequencing run, the sequence quality may be pretty bad. If so, you're clone could be correct and you're getting fooled by bad sequence. You can't assess this parameter doing a pairwise BLAST. If you don't have Sequencher, I would still recommend trying to find some sort of sequence analysis software, there's quite a bit out there for free. Good luck. Wilson ------------------------------------------------------------------------ -------------------- Wilson Clements, Ph.D. wclements@ucsd.edu Dept. of Biology Section of Cell and Developmental Biology University of California at San Diego 9500 Gilman Dr. Natural Sciences Building 6105 La Jolla, CA 92093-0380 TEL (858) 534-6955 LAB (858) 822-4658 FAX (858) 822-5740 Begin forwarded message: > Hi Joanna, > > Thanks for the answer. The strange thing is that I do not get the same > ?errors? when sequencing multiple clones. I can only think that this > is due to the fact that the cDNA was made from multiple larvae > ? Or > that although I used HF the enzyme is causing errors. In any case, > when I use blastx for protein sequence (part of the protein ? 900bp) I > get something like this: > Score = 446 bits (1147), Expect = 1e-123 > Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221 > (0%) > Frame = +2 > > (One alanine substituting a threonine) > Is such a substitution a problem? > Thanks very much > E.A. > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080423/c2159b98/attachment.html From Christian.Broesamle from med.uni-muenchen.de Thu Apr 24 02:29:55 2008 From: Christian.Broesamle from med.uni-muenchen.de (Christian.Broesamle@med.uni-muenchen.de) Date: Thu Apr 24 10:49:22 2008 Subject: [Zbrafish] Re: Sequence homology for protein expression - help! In-Reply-To: <3985ceeb-43e5-4ec6-8041-1715c6c8f01d@m44g2000hsc.googlegroups.com> References: <3985ceeb-43e5-4ec6-8041-1715c6c8f01d@m44g2000hsc.googlegroups.com> Message-ID: <63063.91.12.167.1.1209022195.squirrel@wifomail.med.uni-muenchen.de> Another possibility would be sequencing errors. Did you look at the sequencing electropherogram (the colorful curves thing) to assess the quality of your sequence that usually deteriorates towards the end of the sequencing run and in particular at the positions in question? As to whether the T to A substitution is affecting function, I think there is no safe way to predict. There are some indications such as cross-species conservation of the particular residue, location in a defined and conserved functional domain or active site (phosphorylation site), that would raise my concerns. But in the end, I don't think would want to take the chances. What if your experiment doesn't work? You'd have to go back and reclone and start all over again. Is there a possibility you could order an EST with the right sequence? You could PCR from this and likely get the right sequence, because you start with A LOT of correct template and need fewer cycles (or even clone by restriction digest). Christian > > Hi Joanna, > > Thanks for the answer. The strange thing is that I do not get the same > ?errors? when sequencing multiple clones. I can only think that this > is due to the fact that the cDNA was made from multiple larvae ? Or > that although I used HF the enzyme is causing errors. In any case, > when I use blastx for protein sequence (part of the protein ? 900bp) I > get something like this: > Score = 446 bits (1147), Expect = 1e-123 > Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221 > (0%) > Frame = +2 > > (One alanine substituting a threonine) > Is such a substitution a problem? > Thanks very much > E.A. > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > From dolores.bonaparte from gmail.com Thu Apr 24 05:43:48 2008 From: dolores.bonaparte from gmail.com (Doloresbf) Date: Thu Apr 24 10:49:47 2008 Subject: [Zbrafish] survival blood collection? Message-ID: <837caaf6-f2a0-4f3c-a88d-3020c87c4490@c65g2000hsa.googlegroups.com> Hi, Are you familiar with survival procedures for blood collection on ZFish? Any help would be much appreciated! Thanks very much. Best. Dolores ................................................ Dolores Bonaparte, DVM Instituto de Medicina Molecular Animal Facility Av. Prof. Egas Moniz, Edif. Egas Moniz 1649-028 Lisboa Portugal From davidstachura from gmail.com Thu Apr 24 12:49:50 2008 From: davidstachura from gmail.com (david stachura) Date: Thu Apr 24 12:55:27 2008 Subject: [Zbrafish] Re: blood collection... In-Reply-To: <200804241704.m3OH4WT18482@net.bio.net> References: <200804241704.m3OH4WT18482@net.bio.net> Message-ID: <6075D9E7-4B12-4FFF-AEBD-6D14AA823AA5@gmail.com> > what do you mean when you say "survival procedures?" do you mean "how can i keep the blood cells alive?" or "how do i keep the fish alive after taking blood?" they don't have much blood (maybe 20-50ul), so it's pretty hard to take blood and have the fish survive (when they are adults). however, i am sure it can be done. i would try to do a cardiac puncture with a hamilton syringe. but i guarantee the survival rates are going to be low. we have tried injecting cells into the heart, and maybe kill 1:5 fish. David Stachura, Ph.D. Cell and Developmental Biology University of California at San Diego 9500 Gilman Dr. Natural Sciences Building 6105 La Jolla, CA 92093-0380 davidstachura@gmail.com dstachura@ucsd.edu http://www.biology.ucsd.edu/labs/traver > ------------------------------ > > Message: 3 > Date: Thu, 24 Apr 2008 03:43:48 -0700 (PDT) > From: Doloresbf > Subject: [Zbrafish] survival blood collection? > To: bionet-organisms-zebrafish@moderators.isc.org > Message-ID: > <837caaf6-f2a0-4f3c-a88d-3020c87c4490@c65g2000hsa.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > > Are you familiar with survival procedures for blood collection on > ZFish? Any help would be much appreciated! > Thanks very much. Best. Dolores > ................................................ > Dolores Bonaparte, DVM > Instituto de Medicina Molecular > Animal Facility > Av. Prof. Egas Moniz, Edif. Egas Moniz > 1649-028 Lisboa > Portugal > > > > ------------------------------ > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish > > End of Zbrafish Digest, Vol 35, Issue 15 > **************************************** From rburdine from Princeton.EDU Tue Apr 29 09:37:32 2008 From: rburdine from Princeton.EDU (Burdine, Rebecca D) Date: Tue Apr 29 11:27:38 2008 Subject: [Zbrafish] Boy problems.... In-Reply-To: <6075D9E7-4B12-4FFF-AEBD-6D14AA823AA5@gmail.com> References: <200804241704.m3OH4WT18482@net.bio.net> <6075D9E7-4B12-4FFF-AEBD-6D14AA823AA5@gmail.com> Message-ID: <04AF14C4EF3CF34682460FB1FDBA98C00F1212@MBCLUSTER.pu.win.princeton.edu> Hi everyone, We have had an interesting problem that I am hoping people might have some insight on. Whether we setup fish in batch breeding inserts or in individual tanks, we get a lot of infertility. The eggs themselves look great, they have good color, clearly undergo cytoplasmic transport, but they aren't fertilized. Our females will sometime slay clutches of up to 200 eggs which 80-100% may be unfertilized. This does vary a bit strain to strain (Tu is the worst, our homemade PWT strain has the least issues). I thought maybe something happened in the last generation of males as they grew up, but we are seeing this in the newest generation too (fish are 6 months to 1 year of age). We are seeing it in strains throughout the facility. My assumption is that the males aren't doing their job even though they look extremely healthy. Does anyone have any ideas? (Hopefully this won't come down to the lack of foot baths. 8) Thanks, 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 From castrand from mail.nih.gov Tue Apr 29 12:18:05 2008 From: castrand from mail.nih.gov (Daniel Castranova) Date: Tue Apr 29 12:26:42 2008 Subject: [Zbrafish] Boy problems.... In-Reply-To: <04AF14C4EF3CF34682460FB1FDBA98C00F1212@MBCLUSTER.pu.win.princeton.edu> References: <200804241704.m3OH4WT18482@net.bio.net> <6075D9E7-4B12-4FFF-AEBD-6D14AA823AA5@gmail.com> <04AF14C4EF3CF34682460FB1FDBA98C00F1212@MBCLUSTER.pu.win.princeton.edu> Message-ID: <0E543754-29E4-4F96-A574-09D239754BF1@mail.nih.gov> Becky, One possibility is poor genetic health (lack of out-crossing). When we used the SJD mapping line, the only way that we could get embryos was by doing IVF. If I set them up naturally they would spawn, but we would get very poor fertilization. You may want to try IVF and see if the males have sperm and they are just not releasing it, or if they are dried up. This answer may explain why results aren't better in the next generation, and why there is line to line variability. Just one possibility. good luck. Dan Daniel Castranova Aquatic Specialist Charles River Laboratories - Contractor Unit on Vertebrate Organogenesis Laboratory of Molecular Genetics, NICHD, NIH Building 6B, Room 322, 6 Center Drive Bethesda, MD 20892 (301)594-0904 castrand@mail.nih.gov On Apr 29, 2008, at 10:37 AM, Burdine, Rebecca D wrote: > > Hi everyone, > > We have had an interesting problem that I am hoping people might have > some insight on. > > Whether we setup fish in batch breeding inserts or in individual > tanks, > we get a lot of infertility. The eggs themselves look great, they > have > good color, clearly undergo cytoplasmic transport, but they aren't > fertilized. > > Our females will sometime slay clutches of up to 200 eggs which > 80-100% > may be unfertilized. > > This does vary a bit strain to strain (Tu is the worst, our > homemade PWT > strain has the least issues). I thought maybe something happened > in the > last generation of males as they grew up, but we are seeing this in > the > newest generation too (fish are 6 months to 1 year of age). We are > seeing it in strains throughout the facility. > > My assumption is that the males aren't doing their job even though > they > look extremely healthy. > > Does anyone have any ideas? > > (Hopefully this won't come down to the lack of foot baths. 8) > > Thanks, > 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 > > > _______________________________________________ > Zbrafish mailing list > Zbrafish@net.bio.net > http://www.bio.net/biomail/listinfo/zbrafish -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/zbrafish/attachments/20080429/81d73a2e/attachment.html