[Zbrafish] Rescue mRNA (reply to Sami)

[Jon D.Moulton]

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
     GENE TOOLS, LLC
     jmoulton at gene-tools.com
     www.gene-tools.com
     (541) 929-7840 x1201