[Zbrafish] Fwd: Zbrafish Digest, Vol 45, Issue 16

Wilson Clements via zbrafish%40net.bio.net (by wclements from ucsd.edu)
Fri Feb 27 14:16:00 EST 2009


Dear Jonathan,

We contemplated the strategy you suggest, i.e. making a bicistronic  
transgene with cmlc2:gfp as a transgenesis reporter.  We abandoned  
this strategy because we felt there was a high danger of crosstalk  
between the two promoter drivers.  In your case, the danger would be  
cmlc2 driving your oncogene and mitfa driving eGfp.  So if you don't  
care if your oncogene might get expressed in the heart, then I guess  
you wouldn't have to worry.

I wasn't sure from your posting why you don't think you will be able  
to tell if the F1 are transgenic without cancer.  I mean the mitfa  
promoter is active from late somitogenesis forward, so it seems to me  
you should be able to see fluorescence if you have a transgenic.   
Perhaps you are trying to do this by "transient transgenesis", which  
is to say looking at effects in the injected generation.  If so, my  
main concern would be neoplastic effects downstream of ectopic  
expression.  We see a lot of ectopic (especially somitic) expression  
of transgenes in the injected generation when using the Tol2 system,  
even when we get nice, clean expression of the transgene in the F1  
stables.

Best,
Wilson
--------------------------------------------------------------------------------------------
Wilson Clements, Ph.D.

wclements from 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:

> From: zbrafish-request from oat.bio.indiana.edu
> Date: February 27, 2009 9:03:32 AM PST
> To: zbrafish from magpie.bio.indiana.edu
> Subject: Zbrafish Digest, Vol 45, Issue 16
> Reply-To: zbrafish from oat.bio.indiana.edu
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> Today's Topics:
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>   1. Zfish injection/integration control (Jonathan Schumacher)
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 26 Feb 2009 11:10:10 -0800 (PST)
> From: Jonathan Schumacher <jaschumac from gmail.com>
> Subject: [Zbrafish] Zfish injection/integration control
> To: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID:
> 	<ea171c1d-a725-4f94-9b99-c507f3a7fbd3 from r22g2000vbp.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Greetings,
>
> I have designed a construct of MITFA driving my oncogene and
> cytoplasmic-expressed EGFP, subsequently cloned into the pDest
> Tol2pA.  We suspect that it will take at least 8 weeks to see whether
> the fish develop cancer and after setting up injections, I realized
> that I have no injection/integration control.  My question is would
> you suggest recloning my construct into the pDest Tol2pA with cmlc2-
> driven EGFP?  I believe that this would lead to double-EGFP labeling
> and am unclear if this would be problematic.  Is there an alternative
> strategy, like coinjecting my destination vector with a cherry-labeled
> cmlc2 (or other)?
>
> Your thoughts & suggestions are greatly appreciated.
>
> Best,
>
> Jonathan
>
>
>
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> End of Zbrafish Digest, Vol 45, Issue 16
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