May 20, 2019;
Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis.
It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors'' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.
innate immune response
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Figure 1. Expression of Innate Immune Response Genes in X. tropicalis and X. laevis RNA-Seq Datasets
(A) Fold change in induction caused by the tbxt/tbxt2 MOs and the control MOs.
(B) Fold change caused by control MO in biological replicates at stages 10 and 36 using RT-qPCR.
(C and D) Fold-change induction of innate immune response genes in X. tropicalis (C) and X. laevis (D) datasets.
Figure 2. Expression of GO-Identified Innate Immune Response Genes in X. tropicalis and X. laevis RNA-Seq Datasets
(A–C) Fold-change expression of innate immune response genes across 29 datasets in X. tropicalis (A), across 13 datasets in X. laevis (B), and in 12 of the tbxt/t2 MO datasets (C). The gray region indicates fold change of <1.5×. Green asterisk (∗) indicates a t test p value of <0.01.
Figure 3. Expression of Literature-Identified Innate Immune Response Genes in X. tropicalis and X. laevis RNA-Seq Datasets
(A–C) Fold-change expression of innate immune response genes across 29 datasets in X. tropicalis (A), 13 datasets in X. laevis (B), and in 12 of the tbxt/t2 MO datasets (C). Gray region indicates fold change of < 1.5×. Green asterisk (∗) indicates a t test p value of < 0.01.
Figure 4. Specific Induction of Innate Immune Response Genes
(A and B) Fold-change expression of genes, which were identified to be significantly activated in the X. tropicalis datasets in both the GO-identified (A) and the literature-identified (B) cohort of genes.
(C–E) Fold-change expression of X. laevis genes ptafr.L/gene13059 (C), socs3.L/gene3766 (D), and socs3.S/gene50103 (E), which were identified to be significantly activated. We used the criteria p value < 0.01 and fold change > 1.5 to define significant.