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Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus
journal contributionposted on 2020-07-17, 16:31 authored by George E Gentsch, Thomas Spruce, Rita S Monteiro, Nick DL Owens, Stephen R Martin, James C Smith
Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.
BrachyuryGC contentTALENXenopusdosageimmune responsemorpholinonull mutationoff-targetsplicingAlternative SplicingAnimalsEmbryo, NonmammalianFemaleGene Expression Regulation, DevelopmentalGene Knockdown TechniquesMesodermMorpholinosOligonucleotides, AntisenseXenopus ProteinsXenopus laevisSmith FC001157SBAS-ackBRF-ack06 Biological Sciences11 Medical and Health SciencesDevelopmental Biology