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Antibody validation by Morpholino

Here is an example of using Morpholinos for antibody validation.

Ratzan W, Rayaprolu V, Killian SE, Bradley R, Kohout SC. The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells. PLoS ONE. 2019;14(4):e0209056. doi:10.1371/journal.pone.0209056

Paper: PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components

Another brick pulled from the wall concealing the mechanism of genetic compensation. Nonsense-mediated decay appears again.

Ma Z, Zhu P, Shi H, Guo L, Zhang Q, Chen Y, Chen S, Zhang Z, Peng J, Chen J. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature. 2019;[Epub ahead of print] doi:10.1038/s41586-019-1057-y

Antisense delivery enhancement strategies: meta-analysis

Comparison of enhancements to antisense delivery (peptides, dendrimers, etc.)

Assisted delivery of antisense therapeutics in animal models of heritable neurodegenerative and neuromuscular disorders: a systematic review and meta-analysis.

van der Bent ML, Paulino da Silva Filho O, van Luijk J, Brock R, Wansink DG.

Sci Rep. 2018 Mar 8;8(1):4181. doi: 10.1038/s41598-018-22316-7.

Photo-Morpholinos: no longer a displayed product line

Important update: as of March 2021, Gene Tools is no longer manufacturing Photo-Morpholino oligos.

The following text was published in February 2020.

We are removing Photo-Morpholinos as a displayed product line. Photo-Morpholinos are hard to use - we have made many but they have led to few publications. Any Photo-Morpholino project involves far more interation and development time than a simple Morpholino knockdown; there are many parameters that need to be optimized and several Photo-Morpholino sequences will likely need to be tested. Labs have ordered them without the funding needed to purchase multiple oligos to fine-tune the system sufficiently to get good data; we end up with disgruntled customers.

The old Photo-Morpholino page is here for reference.
Update March 2021: the Photo-Morpholino page has been reduced to its citation list.

Letter to Editor: Morpholino-Mediated Gene knockdown in Zebrafish: It's All About Dosage and Validation

Morpholino-Mediated Gene knockdown in Zebrafish: It's All About Dosage and Validation.

Van Gils M, Vanakker OM.

J Invest Dermatol. 2019 Jan 29. pii: S0022-202X(19)30042-9. doi: 10.1016/j.jid.2019.01.017. [Epub ahead of print] No abstract available.

This is a discussion of specificity and p53-mediated effects in the context of a reply to a commentary on a recent paper.

Essay: "Knockdowns versus knockouts: why the phenotypic difference?"

Here is an essay by Dani Wijesinghe entitled "Knockdowns versus knockouts: why the phenotypic difference?" from 2016.

Her last line: "No matter how blinding it may be to the current genetic reductionist paradigm, to close curtains on one of the few windows through which light is coming is to leave us in the dark."

Knockdown causing stress response (Lai et al. 2018): my response to a good study

In the Lai et al. 2018 preprint from Didier Stainier's group, Morpholino knockdown of vegfaa showed no stress gene response. This demonstrates that the stress gene upgregulation seen with knockdown of egfl7 and some other transcripts is not a response to the Morpholino backbone but a response to the loss of the target's expression. The Robu et al. 2007 p53 paper showed that if Morpholino knockdown of a transcript caused a p53 response, knockdown of that target with a different oligo type (in their case a modified PNA) caused a similar p53 response, again a response caused by the loss of particular proteins.

(this paragraph added 13 Dec 18) Didier Stainier has a recent preprint describing triggering genetic compensation by RNA fragments from the nonsense-mediated decay pathway. It is possible that it is not the loss of a protein but instead is the product of RNA decay that acts as a trigger for the p53 or stress responses as well, though the preprint describes only the compensation response to RNA fragments. (Genetic compensation is triggered by mutant mRNA degradation. bioRxive. 2018. doi:10.1101/328153)

These studies reveal more about biological responses to a knockdown and the contrast of knockdowns and knockouts. Especially combined with observations of a mutant, the loss of target function in a wild-type organism (an uncompensated background) can reveal more information about the target protein's function and the cellular response to its loss.

As demonstrated by the stress response to the Standard Control oligo at elevated doses, keeping the dose of a Morpholino as low as practicable improves the oligo specificity, decreasing the probability of stress responses.
Lai JKH, Gagalova KK, Kuenne C, El-Brolosy MA, Stainier DYR. Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.Dev Biol. 2019 Oct 1;454(1):21-28. doi: 10.1016/j.ydbio.2019.06.008. Epub 2019 Jun 12
(Lai et al. 2018.

Robu et al. 2007.

Phosphorothioates, Morpholinos and cellular changes

Here is a discussion of protein interactions and other cellular changes caused by phosphorothioate oligos that are not seen with Morpholinos.

Flynn LL, Li R, Aung-Htut MT, Pitout IL, Cooper J, Hubbard A, Griffiths L, Bond C, Wilton SD, Fox AH, Fletcher S. Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing. bioRxiv. 2018:446773[Preprint] doi:10.1101/446773


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