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

Developmental cell | 2018

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 RIS Download

Associated grants

  • Agency: Wellcome Trust, United Kingdom
  • Agency: Medical Research Council, United Kingdom
    Id: MC_U117597140
  • Agency: Medical Research Council, United Kingdom
    Id: FC001-157
  • Agency: Cancer Research UK, United Kingdom
    Id: FC001-157

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This is a list of tools and resources that we have found mentioned in this publication.


Addgene (tool)

RRID:SCR_002037

Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

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Bioconductor (tool)

RRID:SCR_006442

Software repository for R packages related to analysis and comprehension of high throughput genomic data. Uses separate set of commands for installation of packages. Software project based on R programming language that provides tools for analysis and comprehension of high throughput genomic data.

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Thermo Fisher Scientific (tool)

RRID:SCR_008452

Commercial vendor and service provider of laboratory reagents and antibodies. Supplier of scientific instrumentation, reagents and consumables, and software services.

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Gene Expression Omnibus (GEO) (tool)

RRID:SCR_007303

Functional genomics data repository supporting MIAME-compliant data submissions. Includes microarray-based experiments measuring the abundance of mRNA, genomic DNA, and protein molecules, as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology. Array- and sequence-based data are accepted. Collection of curated gene expression DataSets, as well as original Series and Platform records. The database can be searched using keywords, organism, DataSet type and authors. DataSet records contain additional resources including cluster tools and differential expression queries.

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DESeq2 (tool)

RRID:SCR_015687

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RRID:SCR_005275

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RRID:SCR_005663

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RRID:SCR_010881

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LIMMA (tool)

RRID:SCR_010943

Software package for the analysis of gene expression microarray data, especially the use of linear models for analyzing designed experiments and the assessment of differential expression.

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htseq-count (tool)

RRID:SCR_011867

Script distributed with the HT-Seq Python framework for processing RNA-seq or DNA-seq data.

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RRID:SCR_012802

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RRID:SCR_005012

Functional genomics data repository supporting MIAME-compliant data submissions. Includes microarray-based experiments measuring the abundance of mRNA, genomic DNA, and protein molecules, as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology. Array- and sequence-based data are accepted. Collection of curated gene expression DataSets, as well as original Series and Platform records. The database can be searched using keywords, organism, DataSet type and authors. DataSet records contain additional resources including cluster tools and differential expression queries.

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Blast2GO (tool)

RRID:SCR_005828

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RRID:SCR_010236

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RRID:SCR_013035

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RRID:SCR_013623

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RRID:SCR_017639

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RRID:SCR_004463

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RRID:SCR_007164

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RRID:SCR_001905

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RRID:SCR_015899

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RRID:SCR_002105

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RRID:SCR_008394

Programming language for all operating systems that lets users work more quickly and integrate their systems more effectively. Often compared to Tcl, Perl, Ruby, Scheme or Java. Some of its key distinguishing features include very clear and readable syntax, strong introspection capabilities, intuitive object orientation, natural expression of procedural code, full modularity, exception-based error handling, high level dynamic data types, extensive standard libraries and third party modules for virtually every task, extensions and modules easily written in C, C (or Java for Python, or .NET languages for IronPython), and embeddable within applications as a scripting interface.

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RRID:AB_228307

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RRID:AB_260581

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RRID:AB_260092

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RRID:AB_260581

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View all literature mentions

Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (antibody)

RRID:AB_228307

This polyclonal secondary targets IgG (H+L)

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RRID:AB_260092

This monoclonal targets HA antibody produced in mouse

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