Rescue mRNA (reply to Sami) (Wilson K. Clements)

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.

Best,
Wilson


------------------------------------------------------------------------
--------------------
Wilson Clements, Ph.D.

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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
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On May 9, 2006, at 10:00 AM, [Only registered users see links. ] wrote: