Morpholinos with specificity controls please
Posted by Jon Moulton, on 16 September 2020
Please, always run specificity controls with Morpholinos. Morpholinos can reveal gene functions that can be concealed by genetic compensation in a mutant, but rigorous specificity controls are essential to determining whether a morphant phenotype is due to knockdown of the oligo target or an unexpected interaction with a different RNA. The Morpholino-in-a-mutant approach described in Jiang et al. 2020 (linked below) is an excellent approach when a mutant is available.
For those using Morpholinos, these three papers provide important perspective on the need for and good design of specificity controls.
On the mechanism behind many differing phenotypes of morphants and mutants:
Sztal TE, Stainier DYR. Transcriptional adaptation: a mechanism underlying genetic robustness. Development. 2020 Aug 14;147(15):dev186452. doi: 10.1242/dev.186452.
https://dev.biologists.org/content/147/15/dev186452
Describing a case where the mutant – morphant phenotypic difference is not caused by transcriptional adaptation:
Jiang Z, Carlantoni C, Allanki S, Ebersberger I, Stainier DYR. Tek/Tie2 is not required for cardiovascular development in zebrafish. Development. 2020 Sep 14:dev.193029. doi: 10.1242/dev.193029.
https://dev.biologists.org/content/early/2020/09/12/dev.193029
On Morpholino controls:
Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB. Guidelines for morpholino use in zebrafish. PLoS Genet. 2017 Oct 19;13(10):e1007000. doi: 10.1371/journal.pgen.1007000. eCollection 2017 Oct.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007000
Thanks,
– Jon
Jon D. Moulton
GENE TOOLS, LLC
(1 votes)
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Since posting the appeal for specificity controls and the citations above, more discussions and methods relevant to Morpholino specificity have appeared.
Zebrafish prrx1a mutants have normal hearts.
https://www.nature.com/articles/s41586-020-2674-1
Reply to: Zebrafish prrx1a mutants have normal hearts
https://www.nature.com/articles/s41586-020-2675-0
Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes.
https://www.nature.com/articles/s41598-020-71708-1