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Dev Cell March 12, 2018; 44 (5): 597-610.e10.
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Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus.

Gentsch GE , Spruce T , Monteiro RS , Owens NDL , Martin SR , Smith JC .

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.

PubMed ID: 29478923
PMC ID: PMC5861998
Article link: Dev Cell
Grant support: [+]

Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: abi1 actc1 bloc1s4 c3ar1 casp10 cav1 cav3.2 cdx1 cdx2 cdx4 chit1 dtymk esr-5 exosc9 foxn4 gdf3 hes7.2 hoxd8 il1b irf1 map3k14 marchf8 meox2 mesp2 mespa msgn1 myc myf5 myh6 myod1 ncf4 pram1 ptbp1 rfx2 ripk3l.2 ripply2.1 ripply2.2 slc22a18 socs3 tal1 tbx18 tbx6 tbxt tbxt.2 tdgf1.3 tf tlr5 tnf tp53 tp53inp1 tra2b tyro3
Antibodies: HA Ab20 Myc Ab12 Tuba4a Ab3
Morpholinos: tbxt MO2 tbxt MO3 tbxt.2 MO3 tbxt.2 MO4

GEO Series: GSE96655: Xenbase,  NCBI

Article Images: [+] show captions
References [+] :
Abu-Daya, Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6. 2010, Pubmed, Xenbase