PUBLICATION
Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes
- Authors
- Cunningham, C.M., Bellipanni, G., Habas, R., Balciunas, D.
- ID
- ZDB-PUB-201002-115
- Date
- 2020
- Source
- Scientific Reports 10: 15366 (Journal)
- Registered Authors
- Balciunas, Darius, Bellipanni, Gianfranco
- Keywords
- none
- MeSH Terms
-
- 5' Untranslated Regions/drug effects
- 5' Untranslated Regions/genetics
- Alleles
- Animals
- Binding Sites/genetics*
- Embryo, Nonmammalian
- Gene Expression Regulation, Developmental/drug effects
- Gene Expression Regulation, Developmental/genetics
- Gene Knockdown Techniques/methods
- Genetic Techniques
- Morpholinos/pharmacology*
- Mutation/drug effects
- Mutation/genetics
- Phenotype
- RNA Splice Sites/drug effects
- RNA Splice Sites/genetics
- Sensitivity and Specificity
- Zebrafish/genetics*
- Zebrafish Proteins/genetics*
- Zygote/drug effects
- PubMed
- 32958829 Full text @ Sci. Rep.
Citation
Cunningham, C.M., Bellipanni, G., Habas, R., Balciunas, D. (2020) Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes. Scientific Reports. 10:15366.
Abstract
Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping