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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

ASO-mediated RNA knockdown correlates with phenotype.A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to ef1a) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of MALAT1: 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 smad2 ASO, 50 pg smad5 ASO, 50 pg alk8 ASO, 50 pg bmp7a ASO, 150 pg chordin ASO, 100 pg ntla ASO, 50 pg wnt11 ASO, 150 pg tolloid ASO, 100 pg wnt5b ASO, 100 pg nacre ASO, 150 pg MALAT1 ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for alk8, bmp7a, chordin, smad5 and ctnnb2 (shown are 3 embryos representative of the different severities of ctnnb2 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) dnd ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg eGFP-nanos3’UTR mRNA. Coinjection of 25 pg dnd ASO caused complete loss of green germ cells (white arrow).
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pone.0139504.g004: ASO-mediated RNA knockdown correlates with phenotype.A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to ef1a) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of MALAT1: 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 smad2 ASO, 50 pg smad5 ASO, 50 pg alk8 ASO, 50 pg bmp7a ASO, 150 pg chordin ASO, 100 pg ntla ASO, 50 pg wnt11 ASO, 150 pg tolloid ASO, 100 pg wnt5b ASO, 100 pg nacre ASO, 150 pg MALAT1 ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for alk8, bmp7a, chordin, smad5 and ctnnb2 (shown are 3 embryos representative of the different severities of ctnnb2 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) dnd ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg eGFP-nanos3’UTR mRNA. Coinjection of 25 pg dnd ASO caused complete loss of green germ cells (white arrow).

Mentions: To test whether ASOs could be used as a general knockdown reagent in zebrafish, we selected an additional 17 embryonically expressed genes with known mutant phenotypes (see Table 1). For 10 of the targeted genes, we assessed whether ASO injection leads to degradation of the target RNA by qPCR. Each ASO was injected at 4 concentrations, and samples were collected at the peak times of the corresponding gene’s expression during the first 36 hours of development to assess target mRNA levels. After monitoring development of the remaining embryos for 24 hours to determine the highest concentration of ASO injection that produced minimal embryonic death, we processed the corresponding samples for qPCR to determine if there was knockdown of the target mRNA. We observed a strong knockdown for alk8, smad5, smad2, chordin and bmp7a, a partial knockdown for ntla and wnt11 and no knockdown for nacre, tolloid and wnt5b (Fig 4A). Notably, knockdown efficiency correlated with phenotype. First, ASOs that caused efficient knockdown of their target mRNAs (e.g. alk8, smad5, smad2, chordin and bmp7a ASOs) reproduced published mutant and knockdown phenotypes in surviving embryos [34–40] (Fig 4B and S2B Fig; quantitation of phenotypes in S2A Fig). Second, 2 of the 3 ASOs that failed to knock down their cognate mRNAs (nacre and tolloid ASOs) did not produce a specific phenotype: they either caused no phenotype or resulted in embryonic deformation and death (S1B Fig; for quantitation of survival and phenotypes see S2A Fig). Results for wnt5b ASO, the third ASO that failed to knock down its target gene based on qPCR, were inconclusive due to high variability and high toxicity (data not shown). Third, ntla and wnt11 ASO injection lead to partial knockdown and reproduced the published mutant phenotype [41] in a smaller proportion of embryos (S2A and S2B Fig). Although we found that in most cases a successful knockdown (measured by qPCR) predicted a loss-of-function phenotype, there was a single case in which ASO knockdown achieved up to 90% reduction in the target mRNA, and yet the injected embryos had few gene-specific phenotypes (oep ASO#1, S2A Fig). To ensure the knockdown was specific to the ASO that was injected, we assessed the level of smad5, bmp7a and alk8 RNA in uninjected embryos and those injected with either a smad5, bmp7a or alk8 ASO. Indeed, only the RNA corresponding to the injected ASO was reduced (Fig 4C). Together, these results suggest that qPCR can be a useful assay to pre-screen ASOs for their ability to knock down target mRNAs.


Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

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

ASO-mediated RNA knockdown correlates with phenotype.A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to ef1a) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of MALAT1: 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 smad2 ASO, 50 pg smad5 ASO, 50 pg alk8 ASO, 50 pg bmp7a ASO, 150 pg chordin ASO, 100 pg ntla ASO, 50 pg wnt11 ASO, 150 pg tolloid ASO, 100 pg wnt5b ASO, 100 pg nacre ASO, 150 pg MALAT1 ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for alk8, bmp7a, chordin, smad5 and ctnnb2 (shown are 3 embryos representative of the different severities of ctnnb2 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) dnd ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg eGFP-nanos3’UTR mRNA. Coinjection of 25 pg dnd ASO caused complete loss of green germ cells (white arrow).
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Related In: Results  -  Collection

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

pone.0139504.g004: ASO-mediated RNA knockdown correlates with phenotype.A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to ef1a) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of MALAT1: 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 smad2 ASO, 50 pg smad5 ASO, 50 pg alk8 ASO, 50 pg bmp7a ASO, 150 pg chordin ASO, 100 pg ntla ASO, 50 pg wnt11 ASO, 150 pg tolloid ASO, 100 pg wnt5b ASO, 100 pg nacre ASO, 150 pg MALAT1 ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for alk8, bmp7a, chordin, smad5 and ctnnb2 (shown are 3 embryos representative of the different severities of ctnnb2 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) dnd ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg eGFP-nanos3’UTR mRNA. Coinjection of 25 pg dnd ASO caused complete loss of green germ cells (white arrow).
Mentions: To test whether ASOs could be used as a general knockdown reagent in zebrafish, we selected an additional 17 embryonically expressed genes with known mutant phenotypes (see Table 1). For 10 of the targeted genes, we assessed whether ASO injection leads to degradation of the target RNA by qPCR. Each ASO was injected at 4 concentrations, and samples were collected at the peak times of the corresponding gene’s expression during the first 36 hours of development to assess target mRNA levels. After monitoring development of the remaining embryos for 24 hours to determine the highest concentration of ASO injection that produced minimal embryonic death, we processed the corresponding samples for qPCR to determine if there was knockdown of the target mRNA. We observed a strong knockdown for alk8, smad5, smad2, chordin and bmp7a, a partial knockdown for ntla and wnt11 and no knockdown for nacre, tolloid and wnt5b (Fig 4A). Notably, knockdown efficiency correlated with phenotype. First, ASOs that caused efficient knockdown of their target mRNAs (e.g. alk8, smad5, smad2, chordin and bmp7a ASOs) reproduced published mutant and knockdown phenotypes in surviving embryos [34–40] (Fig 4B and S2B Fig; quantitation of phenotypes in S2A Fig). Second, 2 of the 3 ASOs that failed to knock down their cognate mRNAs (nacre and tolloid ASOs) did not produce a specific phenotype: they either caused no phenotype or resulted in embryonic deformation and death (S1B Fig; for quantitation of survival and phenotypes see S2A Fig). Results for wnt5b ASO, the third ASO that failed to knock down its target gene based on qPCR, were inconclusive due to high variability and high toxicity (data not shown). Third, ntla and wnt11 ASO injection lead to partial knockdown and reproduced the published mutant phenotype [41] in a smaller proportion of embryos (S2A and S2B Fig). Although we found that in most cases a successful knockdown (measured by qPCR) predicted a loss-of-function phenotype, there was a single case in which ASO knockdown achieved up to 90% reduction in the target mRNA, and yet the injected embryos had few gene-specific phenotypes (oep ASO#1, S2A Fig). To ensure the knockdown was specific to the ASO that was injected, we assessed the level of smad5, bmp7a and alk8 RNA in uninjected embryos and those injected with either a smad5, bmp7a or alk8 ASO. Indeed, only the RNA corresponding to the injected ASO was reduced (Fig 4C). Together, these results suggest that qPCR can be a useful assay to pre-screen ASOs for their ability to knock down target mRNAs.

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