[Zbrafish] Re: morpholino blues

pavlina.haramis from gmail.com via zbrafish%40net.bio.net (by pavlina.haramis from gmail.com)
Wed May 28 03:50:43 EST 2008


On May 27, 9:30 pm, "Burdine, Rebecca D" <rburd... from Princeton.EDU>
wrote:
> Hi Gilbert,
>
> In general I would say 50% or less of the MOs we order work.  I know they don't work because most of the time we have actual mutants we cannot phenocopy. We want the MOs so we can have all the embryos essentially mutant for biochemistry and other experiments.  When we order MOs to knock down genes where we have no idea what the phenotype is, I am always very skeptical.
>
> Some odd results for us include:
> One case where we have a splice morpholino we are 99% sure actually affects maternal.  In another case we have ordered 5 different MO to a gene (3 start site, 2 splice) and only 1 splice MO works sort of.  At least twice we have ordered MOs from publications and can't get them to work either.  When we contact the authors we are told they only work in TL or AB or that we have to really titrate or do something else.  My feeling from this is lots of people have similar issues.
>
> When they work, they are phenomenal. We have some that give great results at 250pg per embryo! When they don't, I mourn my $400+. 8)
>
> I always have Genetools design mine for us so I really believe they are as good as we can predict. However for so many of our genes we don't have a great handle on the 5' end or alternative messages so maybe this is part of the problem.
>
> I'd be interested in hearing other people's experiences too.
>
> 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: rburd... from princeton.edu
> Admin Assistant: Cathy Falk (609) 258-1604
>
> > -----Original Message-----
> > From: zbrafish-boun... from oat.bio.indiana.edu
> > [mailto:zbrafish-boun... from oat.bio.indiana.edu] On Behalf Of
> > Gilbert Weidinger
> > Sent: Friday, May 23, 2008 11:54 AM
> > To: Zbraf... from magpie.bio.indiana.edu
> > Subject: [Zbrafish] morpholino blues
>
> > hi all,
>
> > I wonder whether it's conceivable that some genes cannot be
> > targeted with morpholinos. In many years of using MOs I have
> > come to accept that only about 50% of them work, but now we
> > have a case in the lab where NONE out of 4 MOs targeting the
> > same gene has any effect.
>
> > We have 2 splice blockers: they do not alter splicing as
> > determined by RT-PCR.
> > We have 2 translation blockers: they do not inhibit
> > translation of RNA reporter constructs (5'UTR fused to cherry).
>
> > We inject up to 10µg/µl and use both the RT-PCR assay and the
> > reporter RNAs successfully for other MOs (against other
> > genes). Genetools claims that 70% of all MOs knock down their
> > target...
>
> > Of note is that all 4 MOs are tagged with fluorescein. We
> > don't have a lot of experience with these. Is it possible
> > that they are less efficient than untagged MOs?
>
> > I'd appreciate getting some feedback on whether there are
> > other stories like this out there or whether we are simply
> > exceptionally unlucky.
>
> > thanks
> > gilbert
>
> > -----------------------------------------------------------
> > Gilbert Weidinger, PhD
>
> > Group Leader SFB 655
> > Biotechnology Center & Center for Regenerative Therapies
> > Technical University of Dresden
>
> > Mail address:
>
> > Biotechnology Center
> > Tatzberg 47-51
> > 01307 Dresden
> > Germany
>
> > Tel. office:    0049 351 463 40120
> > Tel. lab:       0049 351 463 40108
> > Tel. secretary: 0049 351 463 40345
> > Fax:            0049 351 463 40348
> > email:          gilbert.weidin... from biotec.tu-dresden.de
> > web:              http://www.biotec.tu-dresden.de/weidinger
>
> > _______________________________________________
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> > Zbraf... from net.bio.net
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Hi Gilbert, Becky,
We too have had some crazy problems with Morpholinos. We also have a
gene that we have 4 MOs against 2 ATG-blocking, 2 splice blocking
One splice-blocking works we can show that on RT PCR ( not a massive
efficiency though 50-50 wt message, wrongly spliced message) and gives
us a phenotype. The ATG-blocking gives us a totally different
phenotype! The rescue does not work and the other two MOs give us
normal embryos ( probably don't work)
We also have a case that we can not phenocopy the mutant by MOs in the
same gene. But we also have a case that when you inject the MO in
hets, then you have the phenotype
( probably maternal protein?)
And the weirdest is that we have 3 MOs against the same gene, the all
give the same phenotype, but we can not show molecularly that they
work ( the splice-blocking does not block splicing... and the rescue
does not work either.....
Actually since we are on this "misery-sharing" thread, i have a
question since almost none of our rescues work. Do people just clone
their gene in the pCS2 or do you put stuff like Kozak sequence etc.
Actually could it be that by adding a Kozak you make things worse in
terms of expresion??
I know it sounds daft but i am getting desperate. We do have the SV40
polyA etc and obviously we only try to rescue splice-blocking MO-
induced phenotypes whenthere are is only 5 bp overlap between the gene
and the exogenous mRNA.
That was such a cathartic experience! Thanks Becky!





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