Dataset Information

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

ABSTRACT: Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

SUBMITTER: Pauli A

PROVIDER: S-EPMC4593562 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

Pauli Andrea A Montague Tessa G TG Lennox Kim A KA Behlke Mark A MA Schier Alexander F AF

PloS one 20151005 10

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-fu ...[more]

PMID: 26436892

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Project description:BackgroundAntisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.ResultsAlthough many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers.ConclusionThe combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.

Project description:Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Dataset Information

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

Publications

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

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