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Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

Pauli A, Montague TG, Lennox KA, Behlke MA, Schier AF - PLoS ONE (2015)

Bottom Line: To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes.ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1.Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Masschusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Overview of knockdown and loss-of-function technologies in zebrafish.A) Antisense oligos (ASOs, red) degrade target RNA, morpholinos (MOs, orange) either block splicing or inhibit translation, and Cas9-sgRNA complexes (blue) create double-strand breaks in DNA leading to genomic alterations. B) ASOs are RNA-DNA hybrid oligonucleotides containing 10 central DNA nucleotides flanked by 5 2’O-Methyl (2’OMe) modified RNA nucleotides on either side (5-10-5 arrangement). Individual nucleotides in the ASO are linked by phosphorothioate bonds to increase stability.
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pone.0139504.g001: Overview of knockdown and loss-of-function technologies in zebrafish.A) Antisense oligos (ASOs, red) degrade target RNA, morpholinos (MOs, orange) either block splicing or inhibit translation, and Cas9-sgRNA complexes (blue) create double-strand breaks in DNA leading to genomic alterations. B) ASOs are RNA-DNA hybrid oligonucleotides containing 10 central DNA nucleotides flanked by 5 2’O-Methyl (2’OMe) modified RNA nucleotides on either side (5-10-5 arrangement). Individual nucleotides in the ASO are linked by phosphorothioate bonds to increase stability.

Mentions: One effective strategy for interrogating gene function is to disrupt the generation of a gene product by knockdown or knockout. Knockout technologies, such as CRISPR/Cas9 and homologous recombination, alter the DNA locus of the gene by either introducing a premature stop codon or removing the entire locus (Fig 1A) [1,2]. Knockdown methods, on the other hand, such as RNAi, siRNAs and modified antisense oligonucleotides [3,4], target the mRNA rather than alter the DNA. While it is most reliable to infer gene function by generating a mutant organism, knockdown reagents can provide a more immediate assessment of gene function and can be used to target gene products without disrupting regulatory DNA elements.


Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

Pauli A, Montague TG, Lennox KA, Behlke MA, Schier AF - PLoS ONE (2015)

Overview of knockdown and loss-of-function technologies in zebrafish.A) Antisense oligos (ASOs, red) degrade target RNA, morpholinos (MOs, orange) either block splicing or inhibit translation, and Cas9-sgRNA complexes (blue) create double-strand breaks in DNA leading to genomic alterations. B) ASOs are RNA-DNA hybrid oligonucleotides containing 10 central DNA nucleotides flanked by 5 2’O-Methyl (2’OMe) modified RNA nucleotides on either side (5-10-5 arrangement). Individual nucleotides in the ASO are linked by phosphorothioate bonds to increase stability.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593562&req=5

pone.0139504.g001: Overview of knockdown and loss-of-function technologies in zebrafish.A) Antisense oligos (ASOs, red) degrade target RNA, morpholinos (MOs, orange) either block splicing or inhibit translation, and Cas9-sgRNA complexes (blue) create double-strand breaks in DNA leading to genomic alterations. B) ASOs are RNA-DNA hybrid oligonucleotides containing 10 central DNA nucleotides flanked by 5 2’O-Methyl (2’OMe) modified RNA nucleotides on either side (5-10-5 arrangement). Individual nucleotides in the ASO are linked by phosphorothioate bonds to increase stability.
Mentions: One effective strategy for interrogating gene function is to disrupt the generation of a gene product by knockdown or knockout. Knockout technologies, such as CRISPR/Cas9 and homologous recombination, alter the DNA locus of the gene by either introducing a premature stop codon or removing the entire locus (Fig 1A) [1,2]. Knockdown methods, on the other hand, such as RNAi, siRNAs and modified antisense oligonucleotides [3,4], target the mRNA rather than alter the DNA. While it is most reliable to infer gene function by generating a mutant organism, knockdown reagents can provide a more immediate assessment of gene function and can be used to target gene products without disrupting regulatory DNA elements.

Bottom Line: To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes.ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1.Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Masschusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus