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splicing MO vs translation MO

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splicing MO vs translation MO

Postby rose9999 » Thu Aug 14, 2008 1:57 am

hi Dear friends,


I order two MO from gene-tool company. With the translation MO, I observed very strong convergence & extension phenotype. However, when i used the splicing MO,(first exon-intron) i could not observe the same phenptype. :cry: What is the possible reason for this?

It is possible that what i observed in translation MO is only unspecific effect of MO?


thanks for you reply in advance
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Re: splicing MO vs translation MO

Postby jonmoulton » Mon Aug 18, 2008 6:19 pm

Hi Rose,

First, it is possible that you are seeing an effect of the MO due to interaction with an off-target RNA. You need to control for this. There are several methods; I prefer the use of a second non-overlapping translation-blocking oligo. If each of the translation blockers, used in separate experiments, produce the same phenotype then this supports the hypothesis that the observed phenotypic change is due to the effect of the Morpholinos on the targeted RNA and not an off-target RNA. The two nonoverlapping oligo can be coinjected in subsequent experiments and due to synergy they can usually be used at lower doses, conserving oligo.

The first exon-intron oligo (e1i1) is most likely to cause insertion of intron 1 into the mature mRNA sequence. You should confirm this by RT-PCR if possible. Look at the intronic sequence to see if there is an in-frame stop codon -- if there is an in-frame stop codon then you can be more confident that insertion of the intron will inactivate the protein's activity.


Let me know how I can help.

- Jon (from Gene Tools)
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Postby rose9999 » Tue Aug 19, 2008 5:44 am

Thanks Jon

Actually I ordered another 5-UTR MO. with high does, like 6-7.5ng ,it also caused CE phenotype. But the does that can cause CE phenotype is much higher than the ATG-MO. With ATG-MO, only 1.5ng already caused very strong CE phenotype. I heard that high does of MO can caused CE phenotype, it is off-target effects, dont know it is true or not. That is why I have to order a splicing MO to make sure the phenotype.

I did RT-PCR ,unfortunately, seemed RT did not work well last time , I am trying again today.
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Re: splicing MO vs translation MO

Postby jonmoulton » Tue Aug 19, 2008 4:22 pm

Hi Rose,

Try the coninjection of the AUG and 5'-UTR Morpholinos at reduced doses. This might being you below the threshold of concentration that causes off-target RNA interaction.

However, I suspect that what you are seeing might not be an off-target interaction but instead is the p53-mediated apoptotic effect. If this is the case, the effect is caused not by the Morpholino directly but by the loss of the target protein, so a reduced dose used when producing an oligo-synergy knockdown won't solve the problem: it is the knockdown, not the oligo, that triggers the p53 pathway. In this case you need to knock down p53 separately. Here's the reference describing this technique:
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.
Note that if your splice Morpholino eliminates protein activity and IF your CE phenotype is a p53 apoptotic effect, you should see the same phenotype from the splice modifier and the translation blockers since the p53 effect is triggered by the loss of the protein.

I'd suggest you start with the coinjection (check the sequences of the oligos to see whether they are cross-complementary). If that doesn't get rid of the CE phenotype, consider using a coinjected p53 Morpholino.
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Postby rose9999 » Wed Aug 20, 2008 1:19 am

Thanks Jon,

Activation of p53 by MO only causes necrosis in brain, it won't affect CE phenotype.

Actually, before ordering the splicing MO, I did p53 MO co-injection experiment. ATG-MO ,with 1.5ng,it caused strong CE phenotype and necrosis in brain. I then co-injected ATG-MO 1.5ng together with p53MO 5ng. Necrosis in brain is rescued by coinjection with p53, however CE phenotype can not be rescue.

I heard that high does of MO can cause CE phenotype, this may be caused by knocking down some other unknown gene rather than p53.

Still, I dont know wheher the CE phenotype I observed with ATG MO and high does of 5'-UTR MO is specific or not.

There are two possibilities, one is the CE phenotype I observed from ATG-MO is off-target interaction. The other possibility is my splicing MO did not work at all.( I injected with 10ng, still I could not see any phenotype.) The CE phenotype I observed from ATG MO is specific.


I did RT-PCR again yesterday, In splice MO injected embryos, except the band as produced in wildtype control ( I supposed it is the spliced one since the size is ok , but need do sequencing to finally confirm ) , I also found an extra band under( not upper ) that spliced band although it is very weak. I am doing direct PCR sequencing now .
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Re: splicing MO vs translation MO

Postby jonmoulton » Wed Aug 20, 2008 4:57 pm

Hi Rose,

Try the coinjection experiment with the 5'-UTR oligo and the ATG oligo. If you are seeing the CE phenotype at reduced doses when the oligos are coinjected, that supports the hypothesis the the phenotype is due to knockdown of the targeted gene and not an off-target RNA. Only if the off-target RNA had sites where both of the oligos could bind would you see the dose-synergy effect on an off-target RNA; while there is some probability of both targets being present in the off-target RNA if the off-target RNA is closely related to the intended target RNA, it is very unlikely to occur if the target RNA and the putative off-target RNA are unrelated sequences.

Nice work eliminating the possibility of the p53 pathway causing the CE phenotype.

Keep in mind that many Morpholino-induced splice modifications produce active proteins, sometimes even copying mRNA produced by normal alternative splicing. To increase the chance that a splice modification will eliminate protein activity, we usually aim at either skipping the exon that encodes the active site or triggering a frameshift that, by introducing a premature stop, triggers nonsense-mediated decay of the transcript.

As for your splicing Morpholino, how much larger is the weak second band that shows up when you splice block -- is the size consistent with insertion of the intron which abuts the targeted splice junction? That is not an unusual outcome, it just means that rather than redirecting splicing across the exon (to remove the exon and both flanking introns) you have eliminated splicing of the intron targeted by the Morpholino (see "splice stopped" in the figure below). If there is an in-frame stop codon in the intronic sequence, this will likely trigger nonsense-mediated decay after one round of translation. If the number of bases in the inserted intron is not evenly divisible by three, the insertion will frameshift downstream sequence. Another possibility is that splicing was redirected to remove only part of the intron, the "cryptic intronic" splice in the figure below; it's harder to determine whether this would cause a frameshift unless you sequence the RT-PCR product.

SplicePossibilities.gif
Possible outcomes of blocking a donor site of an internal exon


Here is a paper describing activation of a cryptic site. In this case, the cryptic site was in the exon, leading to a partial exon deletion. In your case the cryptic site may be on the other side of the Morpholino target, in the intron, and cause a partial intron insertion.
Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis. 2001 Jul;30(3):154-6.

This one is available online:
http://www.gene-tools.com/files/draper_etal.pdf

Let me know how I can help.

- Jon
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Re: splicing MO vs translation MO

Postby rose9999 » Thu Aug 21, 2008 7:51 am

Hi Jon,

Thanks a lot for your reply. I think my splicing MO did not work well.

I purified the band and did the sequence. The sequence is the same as the wildtype, that mean at least some RNA still has been spliced. This may explain why phenotype is not clear in splicing morphants

I am trying the coinjection experiment now.
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Postby rose9999 » Fri Aug 22, 2008 12:55 am

I did the synergetic injection yesterday.
With 1.5ng ATG MO. i observed CE phenotype. WIth 7.5ng UTR MO , i also observed CE phenotype, although not so strong as ATG MO.

Yesterday, I injected 0.5ng ( one third of what i used before) ATG MO ,11% showed CE phenotype,
In coinjection experiment, I injected 0.5ng ATG MO together with 2.5ng UTR MO, 20.5% showed CE phenotype.

In both experiment, the CE phenotype are not so strong as I previously observed from 1.5ng ATG MO. They are similar with phenotype that observed from 7.5ng UTR MO.

Still it is hard to say the phenotype is due to knockdown of the target gene.
Should I change the does of each MO and try again?
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Re: splicing MO vs translation MO

Postby jonmoulton » Fri Aug 22, 2008 3:34 pm

The oligos do appear to potentiate one another, suggesting they are interacting with the same target RNA. Your doses of individual oligos in the coinjection experiment (0.5ng ATG MO together with 2.5ng UTR MO) are down low enough that each sequence should be quite specific (that is, at these low doses they wouldn't tolerate many mispairs and still produce a phenotype by interacting with the mispaired RNA). Two nonoverlapping sequences are producing the CE phenotype and are potentiating one another's effect; based on your experiment, I expect that the phenotype is resulting from the intended knockdown. As for further experiments, you need to convince yourself (and reviewers) that you have a specific knockdown. Exploring a few more dose combinations might be a good start.
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Postby rose9999 » Mon Aug 25, 2008 8:27 am

Thank you very much Jon.

I had another problem actually.

I ordered two 5-mis MO of ATG MO , however one caused embryos death. The other did not cause death and did not show similar CE phenotype as I observed from ATG MO, however the embryos are not normal. it showed curled trunk even when I low down the concentration as 2ng.

For this expeiments, just want to make sure the phenotype is not taget off effects,I already ordered six MOs from gene tool ( one ATG MO, two 5-UTR MO, one splicing MO and two 5-mis MO)

DO you think I should ordered another 5-mis MO ?
I found some people seem use 5-mis as control, however some they just use wildtype as control.
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Re: splicing MO vs translation MO

Postby jonmoulton » Mon Aug 25, 2008 5:47 pm

Hi Rose,

I really do not like the 5-mispair experiment. Too often there are unanticipated interactions with other RNAs and it sounds to me like that is what you are observing. The shortened curled tail phenotype is typical when a knockdown triggers a p53-mediated apoptotic interaction, so likely you are seeing the knockdown of an unanticipated RNA with the loss of that protein triggering the p53 effect.

There are typically three methods used to confirm specificity of Morpholino experiment. These are the five mispair experiment, the mRNA rescue experiment and the two-nonoverlapping-oligo experiment.

The five mispair experiment is a legacy from the early days of antisense (the 1980s) when this was the standard specificity test, insisted on by all reviewers. The problem is that the five-mispair sequence can itself have off-target (e.g. partial mispair) interactions, so in some cases it might take trying several five-mispair oligos to find get one that cleanly phenocopies the targeted oligo at high concentrations and has no phenotype at the lower concentrations where the targeted oligo is just having its effect. That combination of a phenocopy at high dose and nil effect at low dose is the ideal for a five mispair experiment, but it is not always realized. Therefore, I suggest avoiding five mispair oligo if possible (unless the reviewers insist) and using one of the other specificity experiments.

In a simple version of an mRNA rescue experiment, the coding sequence of a gene is fused to a different 5'-UTR and the mRNA is coinjected with the Morpholino targeting the gene with its wild-type 5'-UTR. Ideally, transcription of the rescue mRNA replaces the transcription of the Morpholino-suppressed mRNA and so the protein is made and the phenotype is "rescued" to the wild-type condition. The mRNA rescue experiment is beautiful when it works and, when it works, is an excellent proof of specificity. Unfortunately, for many genes, it cannot work. Depending on the gene, the onset of transcription might have critical timing or position for proper development. Injecting mRNA into single-cell zygotes results in transcript being present throughout the embryo from the earliest stages of development. The presence of this mRNA can cause developmental abnormalities even as it replaces the "missing" mRNA that the Morpholino knocked down. So, the applicability of this experiment depends on the choice of gene and there is always some risk.

The two-nonoverlapping-oligo experiment is my favorite proof of specificity and is widely accepted by the zebrafish community. For this experiment, two Morpholino are made targeting the same RNA (for translation blockers) or the same pre-mRNA (for splice modifiers). If each of the pair of oligos produces the same phenotype when they are used in separate embryos, this supports the hypothesis that the phenotype is due to knockdown of the targeted RNA and is not due to an interaction with an off-target RNA. Typically for a translation blocking pair, one oligo is targeted around the AUG and the other is targeted a bit upstream in the 5'-UTR. For splice blockers, targeting the splice donor site and the splice acceptor site of a single exon is a typical strategy. Besides being less prone to noise from off-target effects than the 5'-UTR experiment and, unlike mRNA rescues, being applicable for most genes, there is another advantage to the two-nonoverlapping-oligo experiment. This is that once you have the pair of oligos, you can do coinjections both to test for synergy and to exploit synergy in order to conserve oligo.

So, to sum up and answer your question directly, I don't suggest you buy another 5-mispair. If you need another Morpholino for proof-of-specificity, I suggest following the nonoverlapping oligo strategy instead. Your odds are better of getting the data you need from a single oligo and the strategy is fairly widely accepted in developmental biology.

Let me know how I can help.

Regards,

- Jon
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Postby rose9999 » Tue Aug 26, 2008 8:26 am

Thank you very much Jon,

your answer are very helpful.
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