Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus

Developmental Cell
George E GentschJames C Smith

Abstract

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.

Citations

May 24, 2018·Diseases·Máté VargaKálmán Tory
Jul 13, 2019·American Journal of Physiology. Heart and Circulatory Physiology·Mark E PepinOmar Wever-Pinzon
Apr 11, 2019·Disease Models & Mechanisms·Alexandria T M Blackburn, Rachel K Miller
Jul 13, 2019·Frontiers in Physiology·Micaela LasserLaura Anne Lowery
Sep 25, 2020·Nature·Federico TessadoriJeroen Bakkers
Jan 29, 2019·Frontiers in Cell and Developmental Biology·Maria Luisa CayuelaMaria Caterina Mione
Oct 7, 2020·Lab Animal·Rebecca Leech, Karuna Sampath
Aug 10, 2020·Developmental Cell·Gopal KushawahMiguel A Moreno-Mateos
Feb 13, 2021·Genesis : the Journal of Genetics and Development·Venkatramanan G Rao, Saurabh S Kulkarni
May 22, 2019·Developmental Cell·Kitt D ParaisoKen W Y Cho
Mar 12, 2021·Developmental Biology·Bridget T Jacques-FrickeLaura S Gammill
Mar 9, 2021·Developmental Biology·Anirban BardhanCharles A Ettensohn
Jun 16, 2019·Developmental Biology·Jason K H LaiDidier Y R Stainier

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Datasets Mentioned

BETA
GSE96655

Methods Mentioned

BETA
transgenic
PCR
immunoprecipitation
RNA-seq
biolayer interferometry
electrophoresis
genotyping
in vitro transcription
PCA
ChIP-Seq

Software Mentioned

Gene Tools
Julia
WebLogo
cMO
edgeR
tophat
Bioconductor
limma
leafcutter
Python

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