Morpholino

About: Morpholino is a research topic. Over the lifetime, 1695 publications have been published within this topic receiving 66989 citations.

Effective targeted gene ‘knockdown’ in zebrafish

Abstract: The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.

Morpholino antisense oligomers: design, preparation, and properties.

Abstract: Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by technical problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. Herein is an overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs.

p53 Activation by Knockdown Technologies

Abstract: Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

In vivo genome editing using a high-efficiency TALEN system

Abstract: The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.