Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish
Publication Date
October 05, 2015
Journal
PLOS ONE
Authors
Andrea Pauli, Tessa G. Montague, Kim A. Lennox, Mark A. Behlke, et al
Volume
10
Issue
10
Pages
e0139504
DOI
https://dx.plos.org/10.1371/journal.pone.0139504
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0139504
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/26436892
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593562
Europe PMC
http://europepmc.org/abstract/MED/26436892
Web of Science
000362499200030
Scopus
84947270324
Mendeley
http://www.mendeley.com/research/antisense-oligonucleotidemediated-transcript-knockdown-zebrafish
Events
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2313994"], "description"=>"<p>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.</p>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565136, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0139504.g001", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_knockdown_and_loss_of_function_technologies_in_zebrafish_/1565136", "title"=>"Overview of knockdown and loss-of-function technologies in zebrafish.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-05 03:06:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/2314069", "https://ndownloader.figshare.com/files/2314071", "https://ndownloader.figshare.com/files/2314072"], "description"=>"<div><p>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 <i>Xenopus</i> 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 <i>oep</i>, <i>chordin</i>, <i>dnd</i>, <i>ctnnb2</i>, <i>bmp7a</i>, <i>alk8</i>, <i>smad2</i> and <i>smad5</i> in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) <i>MALAT1</i>. 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.</p></div>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565155, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0139504.s001", "https://dx.doi.org/10.1371/journal.pone.0139504.s002", "https://dx.doi.org/10.1371/journal.pone.0139504.s003"], "stats"=>{"downloads"=>10, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Antisense_Oligonucleotide_Mediated_Transcript_Knockdown_in_Zebrafish_/1565155", "title"=>"Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-10-05 03:06:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/2314011"], "description"=>"<p>A) Injection of <i>oep</i> ASO induces dosage-dependent <i>oep</i> phenotypes that resemble zygotic (<i>Zoep</i>) and maternal-zygotic (<i>MZoep</i>) <i>oep</i> genetic mutants. B) <i>oep</i> ASO and <i>MALAT1</i> ASO knockdowns are specific. The RNA levels of <i>oep</i> and <i>MALAT1</i> were measured by qPCR in <i>oep</i> ASO (100 pg) and <i>MALAT1</i> ASO (80 pg)-injected embryos. Shown is the fold change in RNA level compared to WT (wildtype), normalized to <i>ef1a</i> (error bars: standard deviation of the mean of 3 independent experiments). C) qPCR-based measurement of <i>oep</i> RNA levels in individual <i>oep</i> ASO (100 pg)-injected (red) or uninjected (black) embryos at shield stage (6 hpf). D) Rescue of <i>oep</i> ASO-induced <i>oep</i> phenotypes by coinjection of an <i>oep</i> ASO-resistant <i>RFP-oep</i> fusion mRNA. Note that the <i>oep</i> ASO-sensitive <i>RFP-oep</i> fusion mRNA is efficiently knocked down (no red fluorescence) and does not rescue. E) Quantitation of survival at 24 hpf and F) quantitation of phenotypic strength in survivors at 24 hpf in the presence versus absence of p53 (<i>p53</i> MO-injected embryos) or <i>RFP-oep</i> fusion mRNA rescue construct. The number of embryos in each category is indicated.</p>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565141, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0139504.g002", "stats"=>{"downloads"=>2, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_and_specificity_of_oep_ASO_induced_oep_one_eyed_pinhead_mutant_phenotypes_/1565141", "title"=>"Efficiency and specificity of <i>oep</i> ASO-induced <i>oep</i> (<i>one-eyed-pinhead</i>) mutant phenotypes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-05 03:06:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/2314014"], "description"=>"<p><i>MALAT1</i> and <i>ef1a</i> RNA levels were measured for 5 days post injection by qPCR in uninjected (= wildtype), <i>MALAT1</i> ASO (100 pg) and <i>dnd</i> ASO (25 pg)-injected embryos. Shown are <i>MALAT1</i> RNA levels normalized to <i>ef1a</i>. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each).</p>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565143, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0139504.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ASO_mediated_RNA_knockdown_persists_for_several_days_/1565143", "title"=>"ASO-mediated RNA knockdown persists for several days.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-05 03:06:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/2314022"], "description"=>"<p>A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to <i>ef1a</i>) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of <i>MALAT1</i>: no mutant phenotype), orange = reproduced published phenotype in a smaller percentage of embryos, red = did not produce a phenotype. Injected amount of ASO per embryo: 50 pg <i>smad2</i> ASO, 50 pg <i>smad5</i> ASO, 50 pg <i>alk8</i> ASO, 50 pg <i>bmp7a</i> ASO, 150 pg <i>chordin</i> ASO, 100 pg <i>ntla</i> ASO, 50 pg <i>wnt11</i> ASO, 150 pg <i>tolloid</i> ASO, 100 pg <i>wnt5b</i> ASO, 100 pg <i>nacre</i> ASO, 150 pg <i>MALAT1</i> ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for <i>alk8</i>, <i>bmp7a</i>, <i>chordin</i>, <i>smad5</i> and <i>ctnnb2</i> (shown are 3 embryos representative of the different severities of <i>ctnnb2</i> ASO-induced phenotypes). C) ASOs only target the cognate RNA, and not unrelated RNAs. qPCR-based assessment of ASO specificity to their cognate target genes (10 embryos each). D) <i>dnd</i> ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg <i>eGFP-nanos3’UTR</i> mRNA. Coinjection of 25 pg <i>dnd</i> ASO caused complete loss of green germ cells (white arrow).</p>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565148, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0139504.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ASO_mediated_RNA_knockdown_correlates_with_phenotype_/1565148", "title"=>"ASO-mediated RNA knockdown correlates with phenotype.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-05 03:06:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/2314023"], "description"=>"<p>*partial knockdown effect</p><p>For list of all ASO sequences, including information regarding effectiveness, see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139504#pone.0139504.s003\" target=\"_blank\">S1 Text</a>.</p><p>Overview of ASO experiments.</p>", "links"=>[], "tags"=>["Mouse Model Systems", "knockdown reagent", "Zebrafish Antisense oligonucleotides", "smad 5", "concentration range", "lncrna", "phenotype", "zebrafish", "mo", "noncoding RNA", "zygotic transcripts", "knockdown efficiency", "tissue culture", "MALAT 1. ASOs", "gene knockdown reagents", "RNase H"], "article_id"=>1565149, "categories"=>["Uncategorised"], "users"=>["Andrea Pauli", "Tessa G. Montague", "Kim A. Lennox", "Mark A. Behlke", "Alexander F. Schier"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0139504.t001", "stats"=>{"downloads"=>4, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_ASO_experiments_/1565149", "title"=>"Overview of ASO experiments.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-10-05 03:06:21"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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