Brief discussion of Morpholino efficacy, specificity & controls from review in J. Cardiovasc. Dev. Dis.
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
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 .
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 . 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.
 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.
 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.
 Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.
 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.