Review
Advances in the Study of Heart Development and Disease Using Zebrafish
Daniel R. Brown, Leigh Ann Samsa, Li Qian, Jiandong Liu.
J. Cardiovasc. Dev. Dis. 2016, 3(2), 13; doi: 10.3390/jcdd3020013
http://www.mdpi.com/2308-3425/3/2/13/htm
Keyword search "Morpholino" or go to section "3.1.2. Morpholinos" for a brief discussion of Morpholino efficacy, specificity and controls.
There are a few points in this discussion (3.1.2. Morpholinos) where I disagree, others where I would like to expand on their suggestions.
First, the authors write that Morpholinos are designed to bind to translation-initiation sites of mRNA. We have made many successful oligos targeting upstream of the translation initiation AUG; Morpholinos targeting the 5'-UTR block the scanning complex from completing its journey from the 5'-cap to the start codon.
The authors claim that Morpholinos are slowly degraded by cellular processes. However, when this was explicitly tested no degradation was found [1,2]. Morpholino activity does decrease with time; we hypothesize that when a Morpholino binds an RNA and the RNA is degraded by nucleases, a footprint of RNA is protected bound to the Morpholino. The slow degradation of the footprint and release of single-stranded Morpholino activity explains the persistent low-level splice-modifying activity observed months after Morpholino dosing [3].
In Box 2, the Specificity section mentions several control Morpholinos. In section 4, they list the standard control, the 5-base pair mismatch, and the p53 oligo. I've comments about each.
Standard control: This is an oligo targeting an intronic site in human beta-globin that is mutated in some cases of beta-thalaslemia. It has been extensively used as a negative control for developmental biology, so extensively that some experienced Morpholino users have suggested there is no reason to ever inject it again. I agree, but results from your reviewers may vary.
Five base pair mismatch: This is an old-style specificity control oligo, but I think a better control for specificity is to use a second non-overlapping Morpholino to phenocopy the first oligo's results. Sometimes there is phenotypic bleed-through with the five-mispair oligo. On the whole, I'd be happier if everyone used the second targeting oligo approach.
p53 oligo: this is a very important control, used to determine whether the p53-mediated apoptotic cascade is triggered by loss of a protein [4]. The Morpholino results in a zebrafish paper are stronger if, along with reporting the outcome on injecting a targeting oligo, the results of a p53 oligo co-injected with the targeting oligo are also reported.
[1] Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.
[2] Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.
[3] Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.
[4] 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.
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