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Storage of Morpholinos: refrigerate or room temperature?

Morpholino oligos are completely chemically & biologically (enzymatically) stable sitting in water at 4C or at room temperature as long as the pH is not extreme. However, they undergo physical changes that can affect their biological activity. Chilled storage increases the tendency of the oligos to associate with container walls (this is especially a problem if the oligos undergo freeze-thaw cycles). Once a Morpholino has associated tightly with the wall of a container, it can be difficult or impossible to get the oligo back into solution at neutral pH. Very acidic conditions can protonate the oligo and get it back into the bulk solution phase, but can also damage the Morpholino’s backbone, usually by hydrolyzing off the dimethylamine; this is useful for assessing the presence of Morpholinos on the walls of a dry tube, but not for recovering biological activity of a stock solution that has underwent wall-association. Because of this, we do not recommend storing Morpholinos at 4C.

At room temperature, wall association occurs less but solution-phase aggregation occurs more frequently. This aggregation can decrease the single-stranded antisense activity of a Morpholino solution. However, the biological activity of the solution can usually be recovered by autoclaving. Faced with a choice between the irrecoverable loss of activity though container wall association at low temperature versus the recoverable loss of activity through aggregation at room temperature, we recommend room temperature storage.

Proposed mechanism underlying genetic compensation

Genetic paradox explained by nonsense. Wilkinson MF. Nature. 2019;568:179-180. doi: 10.1038/d41586-019-00823-5
https://www.nature.com/articles/d41586-019-00823-5

Genetic compensation triggered by mutant mRNA degradation. El-Brolosy MA et al. Nature. 2019;568:193-197. doi:10.1038/s41586-019-1064-z
https://www.nature.com/articles/s41586-019-1064-z

PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Ma Z et al. Nature. 2019;568:259-263. doi:10.1038/s41586-019-1057-y
https://www.nature.com/articles/s41586-019-1057-y

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

https://journals.plos.org/plosone/article?id=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

https://www.nature.com/articles/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.

https://www.nature.com/articles/s41598-018-22316-7

Photo-Morpholinos: no longer a displayed product line

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.
http://www.gene-tools.com/photomorpholinos

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.

https://www.jidonline.org/article/S0022-202X(19)30042-9/pdf

Review of DMD therapies for restoring dystrophin expression

Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches.

Shimizu-Motohashi Y, Komaki H, Motohashi N, Takeda S, Yokota T, Aoki Y.

J Pers Med. 2019 Jan 7;9(1). pii: E1. doi: 10.3390/jpm9010001. Review.

https://www.mdpi.com/2075-4426/9/1/1/htm

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.

https://medium.com/@daniwijesinghe/knockdowns-versus-knockouts-why-the-p...

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."

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