Dear Jon:
here can I ask you about my MO problem.
I have ordered two splicing site MO: i1e2 and e2i2

RT-PCT: e2i2 MOdidn't remove the intron and i1e2 MO show no genetype effection
phenotype: e2i2 shows very specific phenotype of truncated motor neuron axons at 24hpf but always accompany with retarded development. i1e2 show no phenotype.

I confirmed the phenotype is not because of the the retarded development by age matching experiment. 1) There is no such phenotype in control MO retarded embryos. 2)The MO retarded embryos keep the phenotype when developed until 33hpf.
Now there is two possibility left: 1) this is a MO specific effection. 2) This is the MO toxic effection.

do you think it make sense? and how I can know its not a toxic effection? and I cant seperate the phenotype from the development retardation. ( If less MO, there is no retardation and no phenotype as well)
can you give me some advice.
Thanks for your help.

Best wishes
Jane
edinburgh university

 

Hi Jane,

Development is often slowed during a knockdown, that's no surprise.  To show that the e2i2 phenotype is due to knockdown of the targeted RNA and not due to an off-target RNA interaction, you need to do the knockdown with a second oligo targeting the same RNA.  Clearly that's what you were intending for the i1e2 oligo, but it sounds like it didn't work (we estimate about 75% success with untried oligo sequences designed against public database sequences).  So far, it appears you are onto a knockdown phenotype with your e2i2 oligo; however, I am unclear what effect you are seeing by RT-PCR.  We expect an e2i2 oligo to exclude exon 2, so the mature mRNA would have exon 1 directly ligated to exon 3.  You write that the oligo didn't remove the intron -- that's not the result we expect, nor is an intron inclusion a wild-type case.  Did I correctly understand that intron 2 was included in your mature mRNA?

One experiment you can try prior to ordering a different oligo is to coinject your i1e2 and e2i2 oligos to see if the i1e2 oligo makes the e2i2 oligo more potent; dose synergy occurs when two oligos target the same RNA and the observed phenotype is specific for that RNA (Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC.  A primer for morpholino use in zebrafish.  Zebrafish. 2009 Mar;6(1):69-77.).  Sometimes oligos that appear to have no activity on their own can still potentiate a second oligo targeting the same RNA (better still, these both target exon 2).  If the synergy effect is strong enough, you might be able to convince reviewers that this is sufficient proof of specificity (it certainly would strongly support the hypothesis that the observed phenotype is due to knockdown of the targeted RNA and not an off-target RNA interaction).

However, to show that the phenotype is specific for your target RNA, you'll likely need another oligo targeted to a different exon.  Alternatively you might be able to phenocopy the i2e2 oligo using a translation blocker.   You should also consider doing a simultaneous p53 knockdown to prove that your nerve development phenotype is not due to the p53-mediated apoptotic pathway (Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC.  p53 activation by knockdown technologies.  PLoS Genet. 2007 May 25;3(5):e78. Epub 2007 Apr 10. ).



 

Thanks Jon,

I did coinject i1e2 and e2i2 to get some excited results today. Here is the MO summerise:

MO i1e2(1mM): phenotype: NO

            RT-PCR: NO change

MO e2i2(1mM): Phenotype : specific phenotype but with development retardation

                RT-PCR:  Primer in exon1&3: no shorter band (so exon2 not deleted)

                              Primer in intron2 and exon6: band! (so intron2 not removed)

                              No large intron2 band with primer in exon1&3 because intron2 is over 2kb

MO i1e2(0.5Mm)+e2i2(0.5Mm): phenotype: specific without retardation J

                                 RT-PCR: Primer in exon1&3: Wild-type band and shorter band( exon2 deleted)

                                               Primer in intron2 and exon6: band (so intron2 not removed)

                                 Results: different type of transcript with MO mixture injection

Dear Jon, do you think the results make sense now?

Thank you!

 

Hi Jane,

Here's my hypothesis.  If you are correct that the e2i2 oligo alone causes an intron 2 insertion, then it appears that the addition of the i1e2 oligo causes some excision of exon 2, so that two populations of mRNA are produced: one having intron 2 included between exon 2 and 3, and the other with intron 1, exon 2 and intron 2 all spliced out.  A single oligo can sometimes produce a population of two mRNAs due to activation of a cryptic splice site (see the Draper paper in Genesis 2001 for an example of this); in your case it appears that the binding of the e2i2 oligo alone produces one population of mature mRNA and the binding of both oligos produces another population of mature mRNAs, so that when both oligos are used together then two mRNA populations are produced together.  This is a fairly simple explanation, but the actual explanation might be more complex, for example if there is a cryptic splice activation occurring as well.  You have an apparent knockdown and a phenotype, so I suggest the next step might be to phenocopy the morphant with a different oligo.  If you can do that, you don't really need to determine exactly what happened; just show the knockdown of the target RNA by two methods that phenocopy one another.

 

 

Hi, Jon, The new start-codon Morpholino works, even more effective than I expected, around 80%, i am so happy about it. Thanks for your help!
 

Thanks for the update, Jane.  Be sure to try coinjecting your translation blocker with one of your splice-blocking oligos; you might find you can produce a stronger knockdown that way.  You can use the data from individual knockdowns to show specificity, then the co-injection will hopefully let you explore more complete silencing.

 

 

Hi Jane,

You should try your new high-efficacy translation blocker with your splice modifiers to see if you can get some dose synergy.  A cleaner story would be to use a second nonoverlapping translation blocker to compare with your current translation blocker as a means of showing specificity.  However, if your current oligos phenocopy each other that's probably plenty to argue for specificity.  Does the phenotype produced by the e2i2 oligo closely resemble the phenotype produced by the translation blocker?  Seeing the same phenotype from splice modifying oligos and a translation blocker supports the hypothesis that the phenotype is due to knocking down the intended RNA.

I am still a bit concerned about a possible p53 effect, as I described previously.  That nerve phenotype could be due to p53-mediated apotosis resulting from knockdown of your targeted gene; if so, the phenotype is not the immediate result of knocking down the target but is part of a cellular response to loss of the expression of the target RNA.