Controlling morpholino experiments: don't stop making antisense

Eisen, Judith S; Smith, James C.

doi:10.1242/dev.001115

Cited by 555 publications

(512 citation statements)

References 77 publications

(86 reference statements)

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“…This study allowed us to define the loss of function properties of one DEPDC5 missense mutation alongside one nonsense mutation found in epileptic patients in vivo. Furthermore, in this report, we have performed all the necessary controls to validate the phenotype derived by knockdown through antisense morpholino oligonucleotides, including the use of both splice and ATG‐blocking oligonucleotides, phenotypic rescue by the WT human cDNA, and injection of mismatch‐oligo controls 32, 33…”

Section: Discussionmentioning

confidence: 99%

Depdc5 knockdown causes mTOR‐dependent motor hyperactivity in zebrafish

Calbiac

Dabacan²,

Marsan

et al. 2018

Ann Clin Transl Neurol

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Objective DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling.MethodsZebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy‐related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression.ResultsHere we report a zebrafish model of Depdc5 loss‐of‐function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy‐associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein.InterpretationThis study demonstrates that Depdc5 knockdown leads to early‐onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy‐associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1‐dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5‐inherited forms of focal epilepsy.

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Section: Discussionmentioning

confidence: 99%

Depdc5 knockdown causes mTOR‐dependent motor hyperactivity in zebrafish

Calbiac

Dabacan²,

Marsan

et al. 2018

Ann Clin Transl Neurol

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“…Mos (Gene Tools, LLC) were designed and injected into fertilized one-cell embryos, as detailed in Table S1. The specificity and inhibitory efficiency of each Mo were determined as described (14). Optimal Mo concentrations (Tables S1 and S2) were determined on the basis of morphological criteria.…”

Section: Methodsmentioning

confidence: 99%

miR-204 is required for lens and retinal development via Meis2 targeting

Conte

Carrella²,

Avellino³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

153

183

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MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in the regulation of gene expression. The roles of individual miRNAs in controlling vertebrate eye development remain, however, largely unexplored. Here, we show that a single miRNA, miR-204, regulates multiple aspects of eye development in the medaka fish (Oryzias latipes). Morpholino-mediated ablation of miR-204 expression resulted in an eye phenotype characterized by microphthalmia, abnormal lens formation, and altered dorsoventral (D-V) patterning of the retina, which is associated with optic fissure coloboma. Using a variety of in vivo and in vitro approaches, we identified the transcription factor Meis2 as one of the main targets of miR-204 function. We show that, together with altered regulation of the Pax6 pathway, the abnormally elevated levels of Meis2 resulting from miR-204 inactivation are largely responsible for the observed phenotype. These data provide an example of how a specific miRNA can regulate multiple events in eye formation; at the same time, they uncover an as yet unreported function of Meis2 in the specification of D-V patterning of the retina.microRNA | eye development | medaka fish

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“…Xenopus laevis is the standard model, but is allotetraploid and hence less suited for genetic approaches than the diploid Xenopus tropicalis, whose genome sequence has been determined (2). Whereas embryological manipulations and gainof-function experiments are major strengths of Xenopus, reverse genetics is currently limited to the use of antisense reagents that provide transient and often incomplete gene knockdown (3). The ability to introduce targeted, heritable mutations that disrupt gene function has remained elusive.…”

mentioning

confidence: 99%

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Young

Cherone

Doyon

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

127

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The frog Xenopus , an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus .

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Controlling morpholino experiments: don't stop making antisense

Cited by 555 publications

References 77 publications

Depdc5 knockdown causes mTOR‐dependent motor hyperactivity in zebrafish

Depdc5 knockdown causes mTOR‐dependent motor hyperactivity in zebrafish

miR-204 is required for lens and retinal development via Meis2 targeting

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

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