A blog for discussing genetic methods in zebrafish, following up on workshop D1 from the 2nd Strategic Conference of Zebrafish Investigators (Asilomar, 2007).
Discussing zebrafish genetic methods
This blog was originally set up to help plan a workshop held at the 2007 SCZI. During this workshop, it was clear that while there are some powerful new advances in genetic methods for the zebrafish, there are important questions about some existing methods, and several methods (notably homologous recombination and RNAi) which would be great for the field, but do not yet exist.
We've left this blog up as a forum in which people can discuss some of these methods. Below are several blog entries describing particular methods. Please add questions and comments under the appropriate heading.
--Chi-Bin Chien, Koichi Kawakami, Todd Evans, Hazel Sive
Monday, January 22, 2007
Topics suggested for the workshop
This was a place to suggest topics for the workshop. Since this thread is now obsolete, please don't comment here.
13 comments:
Anonymous
said...
What about more sophisticated approaches using morpholinos for LOF? For example, is anyone having success caging them, or otherwise delaying delivery to a later developmental stage?
Do you know anyone is working on homologous recombination in fish? Or what is going on with research on pluripotent stem cells and nuclear transplantation? Is there any recent progress?
We have successfully electroporated morpholinos into both the adult fin and retina and knocked-down the protein of interest. In some cases, depending upon the knocked-down gene, we have been able to generate reproducible morphant phenotypes. If this is of interest to anyone, some of this data will be on the poster that I will present and I can bring additional data if there is interest.
Mike Parsons (Johns Hopkins University) will be giving a talk on the bacterial nitroreductase system. Expression of the nitroreductase in a cell- or tissue-specific manner (using either Gal4-UAS or a cell-specific promoter) and the addition of a specific compound results in the death of the cells that express the nitroreductase.
Has anyone had good success with rescuing knockdowns that effect heart development? We are knocking down an important protein in DNA repair. The full knockdown results in death at the MBT. Both hypomorphs and rescues have defined heart defects and lack red cells. The hearts express cmlc2 and contract erratically but do not loop.
I would like to get a summary of current efforts in enhancer trapping and gene trapping - what vectors should we use; how high is the effieciency; does the trap reflect endogenous gene?
I would be interested in hearing about people's experiences with transgene silencing (over generations). I know Perry Hackett published a paper on the use of insulators in fish a while back but incorporating these into transgene vectors doesn't seem to be common practice. Has anyone made use of any of these insulator elements routinely? Silencing has been an issue with a couple of lines I have generated (I have not generated many) and I suspect with a transgenic line I obtained from ZIRC. Just to be clear, I'm talking about transgenes that once expressed and have become variegated or silenced entirely, not about transgenes that did not express to begin with.
As I heard from other members of the community, many of us using morpholinos have recognized that they are not working as good as they did before. I experienced, that they are more toxic and less competent then they where before comparing my one data with an old charge and a new charge of the same morpholino. Also using published data concerning the concentration of a given morpholino did not work for me, either there is no effect or they are toxic to the embryo. I think we should force gene tools to investigate this problem as the whole zebrafish community, since morpholinos are widely used in our field. Maybe we could discuss this at the end of the session.
I would be interested in hearing if anyone has had success with tet-on/off systems (or other drug inducible systems) in zebrafish. We have a lot of experiments where expression in both tissue specific and temporally controlled ways would be beneficial.
Re Morpholinos - one person at gene tools told me greater than 90% of morpholinos they make work. I guess I must be ordering the wrong ones! 8) Is anyone else still having issues with morpholinos that give no effect or non-specific effects?
13 comments:
What about more sophisticated approaches using morpholinos for LOF? For example, is anyone having success caging them, or otherwise delaying delivery to a later developmental stage?
Do you know anyone is working on homologous recombination in fish? Or what is going on with research on pluripotent stem cells and nuclear transplantation? Is there any recent progress?
We have successfully electroporated morpholinos into both the adult fin and retina and knocked-down the protein of interest. In some cases, depending upon the knocked-down gene, we have been able to generate reproducible morphant phenotypes. If this is of interest to anyone, some of this data will be on the poster that I will present and I can bring additional data if there is interest.
A great resources for the community would be:
a) Drug-inducible gene misexpression systems
b) Drug-mediated, tissue-specific system for killing cells
Does anybody know about progress in these areas?
Jesús Torres
Mike Parsons (Johns Hopkins University) will be giving a talk on the bacterial nitroreductase system. Expression of the nitroreductase in a cell- or tissue-specific manner (using either Gal4-UAS or a cell-specific promoter) and the addition of a specific compound results in the death of the cells that express the nitroreductase.
Has anyone had good success with rescuing knockdowns that effect heart development? We are knocking down an important protein in DNA repair. The full knockdown results in death at the MBT. Both hypomorphs and rescues have defined heart defects and lack red cells. The hearts express cmlc2 and contract erratically but do not loop.
I would like to get a summary of current efforts in enhancer trapping and gene trapping - what vectors should we use; how high is the effieciency; does the trap reflect endogenous gene?
I may partly answer the alex's question. We also need comments from other guys using their own vectors for trapping.
I may partly answer the alex's question. We also need comments from other guys using their own vectors for trapping.
I would be interested in hearing about people's experiences with transgene silencing (over generations). I know Perry Hackett published a paper on the use of insulators in fish a while back but incorporating these into transgene vectors doesn't seem to be common practice. Has anyone made use of any of these insulator elements routinely? Silencing has been an issue with a couple of lines I have generated (I have not generated many) and I suspect with a transgenic line I obtained from ZIRC. Just to be clear, I'm talking about transgenes that once expressed and have become variegated or silenced entirely, not about transgenes that did not express to begin with.
As I heard from other members of the community, many of us using morpholinos have recognized that they are not working as good as they did before. I experienced, that they are more toxic and less competent then they where before comparing my one data with an old charge and a new charge of the same morpholino. Also using published data concerning the concentration of a given morpholino did not work for me, either there is no effect or they are toxic to the embryo.
I think we should force gene tools to investigate this problem as the whole zebrafish community, since morpholinos are widely used in our field.
Maybe we could discuss this at the end of the session.
Jochen Holzschuh
I would be interested in hearing if anyone has had success with tet-on/off systems (or other drug inducible systems) in zebrafish. We have a lot of experiments where expression in both tissue specific and temporally controlled ways would be beneficial.
Re Morpholinos - one person at gene tools told me greater than 90% of morpholinos they make work. I guess I must be ordering the wrong ones! 8) Is anyone else still having issues with morpholinos that give no effect or non-specific effects?
Becky
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