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RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

Andrews OE, Cha DJ, Wei C, Patton JG - Sci Rep (2014)

Bottom Line: We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes.By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame.Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Vanderbilt University, Nashville, TN [2].

ABSTRACT
RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

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Related in: MedlinePlus

Convergent silencing of mCherry.(A) Non-CT and CT constructs were designed using the Tol2 transgenesis system with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription of the mCherry open reading frame (see Supplement for maps and sequence information). All constructs harbored heart-specific GFP (cmlc2-GFP) which enabled identification of transgenic embryos. (B) Widespread expression of mCherry was observed in non-CT embryos (386 out of 386 transgenic embryos), whereas near complete loss of mCherry was observed in CT-mCherry embryos at 2 dpf (370 out of 402 transgenic embryos).
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f1: Convergent silencing of mCherry.(A) Non-CT and CT constructs were designed using the Tol2 transgenesis system with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription of the mCherry open reading frame (see Supplement for maps and sequence information). All constructs harbored heart-specific GFP (cmlc2-GFP) which enabled identification of transgenic embryos. (B) Widespread expression of mCherry was observed in non-CT embryos (386 out of 386 transgenic embryos), whereas near complete loss of mCherry was observed in CT-mCherry embryos at 2 dpf (370 out of 402 transgenic embryos).

Mentions: To determine if we could trigger RNAi-mediated gene silencing in zebrafish via convergent transcription, we created plasmids with transcriptional promoters arranged in either a convergent (CT) or nonconvergent (non-CT) manner. The experimental setup included insertion of open reading frame (ORF) sequences between the two promoters with no 5′ or 3′ UTR sequences. For proof of principle, we first targeted mCherry for rapid visualization of silencing. For the non-CT mCherry construct, the full length zebrafish β-actin promoter was inserted upstream of the mCherry open reading frame (ORF). The CT mCherry construct harbored a β-actin promoter at the 5′ end and an inverted CMV promoter at the 3′ end (Fig. 1A). Transient transgenic animals were created by co-injecting embryos at the 1–2 cell stage with the non-CT mCherry construct and Tol2 transposase mRNA27. As a marker of transgenesis, the cardiac myosin light chain promoter (cmlc2) was fused to GFP driving fluorescent expression in the heart28. As expected, transgenic animals containing the non-CT mCherry construct displayed robust expression of mCherry throughout the entire developing embryo (Fig. 1B). In contrast, there was a striking absence of mCherry in transgenic animals created by injection of the CT mCherry construct (Fig. 1B). Except for a few small puncta and some yolk autofluorescence, the levels of mCherry were dramatically reduced to near zero. The lack of mCherry was not due to the absence of the transgene as readily detectable levels of heart GFP were observed (Fig. 1B). Silencing was robust across multiple injections with undetectable levels of mCherry in greater than 92% of the transgenic CT-mCherry embryos. Nearly ~100% of non-CT mCherry transgenic embryos broadly expressed mCherry. Importantly, crossing of founders to generate F1 and F2 generations showed that silencing of mCherry is stable and continues to be maintained.


RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

Andrews OE, Cha DJ, Wei C, Patton JG - Sci Rep (2014)

Convergent silencing of mCherry.(A) Non-CT and CT constructs were designed using the Tol2 transgenesis system with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription of the mCherry open reading frame (see Supplement for maps and sequence information). All constructs harbored heart-specific GFP (cmlc2-GFP) which enabled identification of transgenic embryos. (B) Widespread expression of mCherry was observed in non-CT embryos (386 out of 386 transgenic embryos), whereas near complete loss of mCherry was observed in CT-mCherry embryos at 2 dpf (370 out of 402 transgenic embryos).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048883&req=5

f1: Convergent silencing of mCherry.(A) Non-CT and CT constructs were designed using the Tol2 transgenesis system with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription of the mCherry open reading frame (see Supplement for maps and sequence information). All constructs harbored heart-specific GFP (cmlc2-GFP) which enabled identification of transgenic embryos. (B) Widespread expression of mCherry was observed in non-CT embryos (386 out of 386 transgenic embryos), whereas near complete loss of mCherry was observed in CT-mCherry embryos at 2 dpf (370 out of 402 transgenic embryos).
Mentions: To determine if we could trigger RNAi-mediated gene silencing in zebrafish via convergent transcription, we created plasmids with transcriptional promoters arranged in either a convergent (CT) or nonconvergent (non-CT) manner. The experimental setup included insertion of open reading frame (ORF) sequences between the two promoters with no 5′ or 3′ UTR sequences. For proof of principle, we first targeted mCherry for rapid visualization of silencing. For the non-CT mCherry construct, the full length zebrafish β-actin promoter was inserted upstream of the mCherry open reading frame (ORF). The CT mCherry construct harbored a β-actin promoter at the 5′ end and an inverted CMV promoter at the 3′ end (Fig. 1A). Transient transgenic animals were created by co-injecting embryos at the 1–2 cell stage with the non-CT mCherry construct and Tol2 transposase mRNA27. As a marker of transgenesis, the cardiac myosin light chain promoter (cmlc2) was fused to GFP driving fluorescent expression in the heart28. As expected, transgenic animals containing the non-CT mCherry construct displayed robust expression of mCherry throughout the entire developing embryo (Fig. 1B). In contrast, there was a striking absence of mCherry in transgenic animals created by injection of the CT mCherry construct (Fig. 1B). Except for a few small puncta and some yolk autofluorescence, the levels of mCherry were dramatically reduced to near zero. The lack of mCherry was not due to the absence of the transgene as readily detectable levels of heart GFP were observed (Fig. 1B). Silencing was robust across multiple injections with undetectable levels of mCherry in greater than 92% of the transgenic CT-mCherry embryos. Nearly ~100% of non-CT mCherry transgenic embryos broadly expressed mCherry. Importantly, crossing of founders to generate F1 and F2 generations showed that silencing of mCherry is stable and continues to be maintained.

Bottom Line: We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes.By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame.Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Vanderbilt University, Nashville, TN [2].

ABSTRACT
RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

Show MeSH
Related in: MedlinePlus