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Jon's Blog

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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.

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]

"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:
"Other" targets: ncRNA, repeated elements, etc.

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
Watch gene expression start to happen in an embryo

Watching dynamics of RNA expression - paper describing a visualization technique.

Movie 1:

Wednesday, August 15, 2018 - 13:23
Morpholino duration of effect

A researcher asked about the half-life of the Morpholino. This is much of my response.

Tuesday, August 14, 2018 - 07:49
Off-target Morpholino interaction reported based on mutant and 6ng dose

A prp1 Morpholino injected into zebrafish with mutated prp1 caused significant phenotype at a dose of 6 ng/embryo but had little effect at lower doses (see Supplemental Figure S13). This is a case where the Morpholino-in-mutant experiment not only detected an off-target interaction of the Morpholino but defined a dose where the effect suddenly became strong. Doses of prp1 Morpholino below that threshold produced phenotype at roughly the rate of the standard control oligo injections.

Tuesday, August 7, 2018 - 10:16
"Consensus guidelines for the use and interpretation of angiogenesis assays" with MO discussion

"Consensus guidelines for the use and interpretation of angiogenesis assays" contains a very good discussion of Morpholinos and specificity. Nowak-Sliwinska et al. extensively cite the Stainier et al. "Guidelines for Morpholino Use in Zebrafish". There is a theme in the Morpholino discussion in Nowak-Sliwinska et al.'s paper with which I disagree. They state "However, the best and generally accepted validation for any MO phenotype is confirmation of the same phenotype in a zebrafish genetic mutant." [1] Is this best, or does it exclude a valuable function of Morpholinos?

Thursday, May 24, 2018 - 12:02
Review (2008): Current perspectives in zebrafish reverse genetics

This is an older review (2008), but has a nice discussion of Morpholinos and controls. Photo-Morpholino and Vivo-Morpholino use in zebrafish are not addressed. The newer strategy of using Morpholinos in CRISPR mutants for specificity control is more recent than this paper, as is the understanding of gene compensation in mutants versus relatively uncompensated phenotypes from knockdowns triggered by Morpholino microinjections.

Monday, May 14, 2018 - 10:33
An i1e2 Morpholino triggering intron 1 retention: unusual splice outcome

Here is an example of an unexpected splice-modifying outcome. An oligo was targeted to i1e2, which would normally be expected to skip exon 2. Instead, the oligo caused retention of intron 1. This is the usual outcome of an e1i1 oligo, but not of an i1e2 oligo. Figure 5B shows the gel and the RNA map:

Friday, May 11, 2018 - 09:46
Splicing mutations in human genetic disorders: Review

This is an open-access review covering the mechanism of eukaryotic RNA splicing and diseases caused by mutations in regions affecting splicing.

Abramowicz A, Gos M. Splicing mutations in human genetic disorders: examples, detection, and confirmation. J Appl Genet. 2018. doi: 10.1007/s13353-018-0444-7

Tuesday, April 24, 2018 - 08:55