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[Zbrafish] Re: Rescue mRNA (reply to Sami) (Wilson K. Clements)

Wilson Clements wclements at ucsd.edu
Tue May 9 13:52:51 EST 2006

Dear Sami,

In terms of rescuing morpholino phenotype by mRNA injection, the  
brief procedure is to make RNA and co-inject it with your  
morpholino.  For making the mRNA, I recommend using a kit to make  
capped mRNA.  We use mMessage mMachine from Ambion.  You get plenty  
of mRNA.  You need to make sure that the open reading frame for your  
gene is in an mRNA expression vector (e.g. CS2+) with an RNA  
polymerase promoter 5' to the start of translation and a  
polyadenylation signal at the 3' end.

Here are some additional considerations:

1) You will be co-injecting your morpholino with the mRNA, so your  
morpholino needs to be resuspended in RNase-free buffer.
2) It is best to co-inject the morpholino in the same solution with  
your mRNA,  because that way you reduce the possibility of having  
different mosaic expression for the morpholino and the RNA.  Also,  
you reduce the total number of injections you have to do.
3) The amount of mRNA to use can be very difficult to find.  It is  
easiest to rescue with RNA that does not, on its own, have a  strong  
overexpression phenotype, because then you can just inject a lot  
(200-400pg).  Otherwise, you may have to spend quite a bit of time  
looking for the right amount.

I will add a couple of things to what Dr. Moulton has contributed below.

1)  There is an additional reason  that a rescue might not work even  
though the morpholino phenotype is real (besides a requirement for  
proper timing of mRNA expression):  many genes require tissue- 
specific expression.  Your overexpression will be ubiquitous.    
Therefore, if overexpression in non-endogenous tissues causes a  
phenotype, you will always get messed up fish.  Of course if you are  
just looking to see restoration of a specific trait, tissue, cell  
type, etc., you maybe able to see rescue in despite of getting an  
overexpression phenotype.
2)  Using two different morpholinos to get the same phenotype is  
definitely a good control suggesting specificity.  I would also  
suggest, using a control morpholino with 5 bp mismatch to show that  
it does not give the same phenotype.  You could also try to correlate  
phenotypic severity with morpholino dose, preferably in a blind  
experiment.  If you are using a splice-blocker morpholino, you can  
further correlate severity of phenotype with the degree of transcript  
alteration by RT-PCR.
3)  I have personally had the best success with rescuing splice- 
blocking morpholino phenotypes.  However, if you are using  
translation-blockers, a rescue strategy in addition to the ones  
outlined by Dr. Moulton is to use mRNA for the orthologous gene from  
a different species.  The success of this strategy depends on the  
orthologous gene behaving the same way in zebrafish, so it is  
probably (but not necessarily) best to use a closer relative (e.g.  
from Xenopus).  It's important to confirm that the target sequence  
for the morpholino is not present in the rescue transcript!

Good luck.


Wilson Clements, Ph.D.

wclements at ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6324
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 534-5457

On May 9, 2006, at 10:00 AM, zbrafish-request at oat.bio.indiana.edu wrote:

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>    1. Rescue mRNA (reply to Sami) (Jon D.Moulton)
> ----------------------------------------------------------------------
> Message: 1
> Date: Mon, 08 May 2006 15:29:19 -0700
> From: "Jon D.Moulton" <jmoulton at gene-tools.com>
> Subject: [Zbrafish] Rescue mRNA (reply to Sami)
> To: bionet-organisms-zebrafish at moderators.isc.org
> Message-ID: <445FC63F.9030103 at gene-tools.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> Good day Sami,
> This is Jon from Gene Tools.  I may be able to help a bit.  I won't
> address your question directly, there are plenty of folks on this  
> forum
> more experienced in expressing mRNA for rescues.  However, I'll offer
> some strategic advice that you might find useful -- or at least that
> might stimulate some conversation about rescues among Morpholino  
> users.
> The mRNA rescue is an excellent proof-of-specificity experiment but  
> will
> not work for some genes.  For this experiment, an mRNA is injected  
> which
> codes for the same protein that the Morpholino oligo knocks down, but
> the rescue mRNA has a modified Morpholino binding site so that the
> Morpholino target is not present on the rescue mRNA.  For many genes,
> injecting the Morpholino alone results in a morphant phenotype, while
> coinjection of the proper concentration of Morpholino and rescue mRNA
> results in a wild-type embryo.  However, the timing of the onset of
> translation is critical for some developmental genes and the early  
> onset
> of translation resulting from co-injection of Morpholino and rescue  
> mRNA
> in the early zygote may mess up the developmental process so that the
> embryos never recapitulate the wild-type phenotype.
> That said, the rescue is a very satisfying control when it is
> successful.  If you are planning on using mRNA rescues, I recommend  
> that
> you have your Morpholino targeted in the 5'-UTR without extending into
> the coding sequence.  Some folks have tried rescuing Morpholinos
> targeted to the coding sequence by taking advantage of the degenerate
> genetic code to design mismatches into their rescue mRNAs which do not
> change the amino acids from those encoded by the endogenous mRNA (e.g.
> wobble mismatches).  While this strategy makes sense it has not in
> practice been reliable -- it is better to start with a 5'-UTR  
> Morpholino
> so that you can retain the original sequence of the coding region and
> use an irrelevant 5'-UTR sequence for your rescue mRNA.
> There is a different strategy for confirming specificity which you
> should be aware of.  This is the two non-overlapping 5'-UTR oligos
> experiment.  Of course, I'd like to make another oligo for you, but
> there is another reason to prefer this experiment over the mRNA  
> rescue;
> the early onset of translation resulting from injection of a rescue  
> mRNA
> may alter development from wild-type patterns, while confirming
> specificity with the non-overlapping 5'-UTR oligos experiment  
> eliminates
> that risk.
> The two non-overlapping 5'-UTR oligos experiment involves comparing  
> the
> phenotype induced by injection of two different oligos targeted to  
> block
> translation of the same mRNA. If both sequences induce the same
> phenotype, that supports the hypothesis that the observed phenotype is
> due to knockdown of the targeted gene. This has become a very commonly
> used test of specificity in the zebrafish community.
> A variant on this experiment is to use a splice blocking Morpholino to
> produce the same phenotype as the translation blocking oligo; while  
> this
> is a nice experiment when it works, it can be difficult to determine
> which exon to target in order to knock down the activity of the  
> protein
> and phenocopy the translation blocker's effect. Yet another  
> variation is
> the two-splice-blocker experiment, in which the same exon is  
> targeted in
> separate experiments by a splice donor blocker and a splice acceptor
> blocker. If both of the oligos when used individually cause a clean  
> exon
> excision, the phenotypes induced by the two oligos should be identical
> and, again, the hypothesis that the phenotype was triggered by  
> specific
> excision of the exon is supported. However, activation of a cryptic
> splice site can confound the experiments using splice-blockers so  
> while
> identical results indicate specific knockdown, dissimilar results  
> do not
> preclude that interaction with the pre-mRNA was specific.
> Let me know how I can help.
> Regards,
>   - Jon
>      Jon D. Moulton, Ph.D.
>      Special Projects and Customer Support
>      jmoulton at gene-tools.com
>      www.gene-tools.com
>      (541) 929-7840 x1201
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