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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."

Thursday, December 6, 2018 - 11:01
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.

Friday, November 30, 2018 - 12:26
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

Tuesday, October 23, 2018 - 12:35
How I really feel about five-mispair oligos

I try to send this message with any designs involving five-mispair oligos. A brief version is farther down.

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** Argument for using a different specificity control instead of the five-mispair

Wednesday, September 26, 2018 - 11:46
Purifying and analyzing Morpholinos by HPLC and detecting Morpholinos in biological samples

HPLC purifications are most effective when Morpholinos are conjugated to a relatively big molecule, such as a peptide (say ca. 200 Daltons or larger), or are conjugated with a net-charged moiety and then the conjugate is purified to remove unreacted oligo. If you are adding a relatively small reactive group and subsequently adding the large molecule, it is probably best to do both additions prior to the purification; that way it is easier to separate the unreacted Morpholino and the activated Morpholino from the conjugate of the Morpholino with the large molecule.

Friday, September 14, 2018 - 07:39
Book: Exon Skipping and Inclusion Therapies

Exon Skipping and Inclusion Therapies
Editors: Toshifumi YokotaRika Maruyama
2018 Humana Press, New York, NY
doi:10.1007/978-1-4939-8651-4
ISBN 978-1-4939-8650-7
https://link.springer.com/book/10.1007/978-1-4939-8651-4#toc

Tuesday, September 4, 2018 - 12:06
Negative control Morpholino oligos

Gene Tools offers several negative controls that are offered at reduced prices or we can synthesize custom oligos to use as negative controls. I suggest, at least initially, using the standard control oligo.

Wednesday, August 22, 2018 - 10:57
This blog has an index of selected posts

I use my blog to keep track of Morpholino information that might be useful to me; that can make it tough to find the more broadly useful posts. Here's a linked list of posts I think might be useful to others.

http://www.gene-tools.com/content/selected-posts-jons-blog

Monday, August 20, 2018 - 14:28
Oocyte microinjection in situ (OMIS): Morpholinos in zebrafish oocytes in the ovary

Wu X, Shen W, Zhang B, Meng A. The genetic program of oocytes can be modified in vivo in the zebrafish ovary. J Mol Cell Biol. 2018 Jul 28. doi: 10.1093/jmcb/mjy044. [Epub ahead of print]

https://academic.oup.com/jmcb/article-lookup/doi/10.1093/jmcb/mjy044

"Furthermore, maternal knockdown of dnmt1 by antisense morpholino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethality prior to segmentation period."

Monday, August 20, 2018 - 13:22
Targeting non-coding RNAs: strategies and what we need to design an oligo

Morpholinos have been used to alter activity of non-coding RNAs. A citation list is here:
http://www.gene-tools.com/node/14474

Targeting non-coding RNAs presents special problems. Ideally, we need to know where on the non-coding RNA an activity is located that we can block. You will need to tell us where you want the oligo targeted and we’ll try to find the best oligo sequence targeting that location.

Monday, August 20, 2018 - 09:19

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