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
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.
Jon D. Moulton, Ph.D.
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