[Zbrafish] RE: Low efficacy of splice-blocking morpholino

Wilson Clements via zbrafish%40net.bio.net (by wclements from ucsd.edu)
Tue Mar 13 12:41:47 EST 2007


Dear Weibin,

With respect to your problems with your splice-blocker morpholinos:   
our anecdotal evidence with respect to splice-blocking and  
translation-blocking morpholinos suggests that they are roughly 40%  
effective, i.e. we have effective blocking of transcription/ 
translation with 2 out of 5 novel morpholinos that we have ordered,  
and 3 out of 5 do little or nothing.  Therefore, one conceivable  
solution to your problem is simply to try blocking a different splice  
junction, or to try blocking the same one differently.

That said, here are a few things I would do in your position to try  
to increase the efficacy of the existing morpholinos.  1) After  
thawing the frozen morpholino, and before injecting, heat the  
morpholino to 65C for 5 minutes, to remove any secondary structure.   
2) Use a higher concentration in a smaller volume:  a 4nl drop is a  
pretty traumatic experience for a 1-cell embryo.  3) Make sure you  
inject the single blastomere of 1-cell embryos.

If, as suggested by Dr. Burdine, the reason you continue to see  
correctly spliced message is that the transcript is maternal, you  
might consider using a translation blocker instead of a splice  
blocker.  In that situation, I guess you will have a more difficult  
time showing the morpholino is effective, unless you have an antibody.

Have you considered that necrosis might be a real, and not a non- 
specific, effect?  I agree that at high doses this effect is a common  
non-specific effect, but is it impossible that your gene is required  
for cell cycle progression, cell division, survival, etc.?

Best,
Wilson Clements
------------------------------------------------------------------------ 
--------------------
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


On Mar 13, 2007, at 10:02 AM, zbrafish-request from oat.bio.indiana.edu  
wrote:

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>    1. RE: Low efficacy of splice-blocking morpholino
>       (Burdine, Rebecca D)
>    2. incubator (k.dewinne)
>
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Mon, 12 Mar 2007 15:42:17 -0400
> From: "Burdine, Rebecca D" <rburdine from Princeton.EDU>
> Subject: RE: [Zbrafish] Low efficacy of splice-blocking morpholino
> To: <weibinz from umich.edu>, <Zbrafish from magpie.bio.indiana.edu>
> Message-ID:
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> <9A199EA7817D8844955074BB433A74A52386BC from MBCLUSTER.pu.win.princeton.edu 
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>
> There may be maternal RNA in your embryos which your splice-blocking
> morpholinos may not affect.
>
> You don't say if you get a phenotype or not?  If you do, perhaps  
> you can
> use both morpholinos together at lower doses to enhance the  
> penetrance.
>
> If you aren't getting a phenotype, you may not be able to induce  
> one or
> you may need to knockdown maternal message with an ATG or UTR
> morpholino.
>
> 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 from princeton.edu
> Admin Assistant: Cathy Falk (609) 258-1604
>
> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu
> [mailto:zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of
> weibinz from umich.edu
> Sent: Saturday, March 10, 2007 5:39 PM
> To: Zbrafish from magpie.bio.indiana.edu
> Subject: [Zbrafish] Low efficacy of splice-blocking morpholino
>
> Hi,
> I am testing two splicing -blocking morpholino rencently. I found the
> efficacy of blocking splicing is not high enough. I always detected  
> the
> normaly processed mRNA in the embryos injected with morpholino, in the
> presence of the abberently processed mRNA due to the morpholino. My
> injected amount of morpholino is estimated to be about 16ng per
> zebrafish
> embryo, which I think has hit the upper limit and some embryos( about
> 10%)
> already showed massive necrosis. I used either Danieau's or water to
> dilute
> the morpholino to 0.5mM concentration and inject 2-4 nl per embryo.  
> I am
>
> wondering whether anybody could share with me some tips to increase  
> the
> efficacy.
>
> Thanks a lot.
>
> weibin
>
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> ------------------------------
>
> Message: 2
> Date: 13 Mar 2007 09:45:09 -0700
> From: "k.dewinne" <dewinne_k from yahoo.co.uk>
> Subject: [Zbrafish] incubator
> To: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID: <1173804309.369904.154790 from s48g2000cws.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Dear all,
>
> At the moment I'm trying to get an incubator for the zebrafish eggs to
> hatch under controlled conditions. We want to apply different
> stressors to the eggs, so It's important that at least different light
> and temperature regimes can be applied to the eggs. I was hoping
> somebody could suggest me an incubator that answers to these
> demands.
>
> kind regards!
>
>
>
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> End of Zbrafish Digest, Vol 22, Issue 7
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