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In vivo testing of microRNA-mediated gene knockdown in zebrafish.

Leong IU, Lan CC, Skinner JR, Shelling AN, Love DR - J. Biomed. Biotechnol. (2012)

Bottom Line: The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans.The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest.In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT
The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

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miRNA constructs and their effects in zebrafish embryos. (a) Schematic of the miRNA and the eGFP sensor vectors showing the miRNA target sites as well as the qRT-PCR primer locations. The miRNA vector contains a CMV promoter for ubiquitous expression in all tissues, and a DsRed Express reporter gene and the pre-miRNAs. The pre-miRNAs can be single (only one miRNA) or chained (containing more than one miRNA under the same promoter). The eGPF sensor miRNA also contains a CMV promoter, an eGFP reporter gene, and the mature miRNA target at the 3′ UTR of the eGFP gene. The miRNA target sites for zerg-2 are located in exons 9 (miRNA 1), 14 no. 1 (miRNA 2), 14 no. 2 (miRNA 3), and 3′ UTR (miRNA 4). The miRNA targets for zerg-3 are located in exon 8 (miRNA 1), exon 15 (miRNA 2), and two targets located on the 3′ UTR. (b) The injection of eGFP sensor mRNA alone produces embryos that exhibit green fluorescence only, and embryos injected with both eGFP sensor and miRNA mRNA exhibit both green and red fluorescence. (c) An example of embryos injected with either miRNA with eGFP sensor or scrambled miRNA (negative control) with eGFP sensor. The ∗ indicates the presence of the midbrain hindbrain boundary.
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fig1: miRNA constructs and their effects in zebrafish embryos. (a) Schematic of the miRNA and the eGFP sensor vectors showing the miRNA target sites as well as the qRT-PCR primer locations. The miRNA vector contains a CMV promoter for ubiquitous expression in all tissues, and a DsRed Express reporter gene and the pre-miRNAs. The pre-miRNAs can be single (only one miRNA) or chained (containing more than one miRNA under the same promoter). The eGPF sensor miRNA also contains a CMV promoter, an eGFP reporter gene, and the mature miRNA target at the 3′ UTR of the eGFP gene. The miRNA target sites for zerg-2 are located in exons 9 (miRNA 1), 14 no. 1 (miRNA 2), 14 no. 2 (miRNA 3), and 3′ UTR (miRNA 4). The miRNA targets for zerg-3 are located in exon 8 (miRNA 1), exon 15 (miRNA 2), and two targets located on the 3′ UTR. (b) The injection of eGFP sensor mRNA alone produces embryos that exhibit green fluorescence only, and embryos injected with both eGFP sensor and miRNA mRNA exhibit both green and red fluorescence. (c) An example of embryos injected with either miRNA with eGFP sensor or scrambled miRNA (negative control) with eGFP sensor. The ∗ indicates the presence of the midbrain hindbrain boundary.

Mentions: As the pCS2+ vector does not contain an eGFP reporter gene, one had to be inserted into the vector before the eGFP sensor vector could be used. The Kozak sequence and the eGFP coding region was excised from pT2AL200R150G (a gift from Dr Koichi Kawakami, National Institute of Genetics) using BamHI and ClaI restriction enzymes. The pCS2+ vector was restriction enzyme digested with BamHI and ClaI and the eGFP coding region was cloned into the linearised vector (Figure 1).


In vivo testing of microRNA-mediated gene knockdown in zebrafish.

Leong IU, Lan CC, Skinner JR, Shelling AN, Love DR - J. Biomed. Biotechnol. (2012)

miRNA constructs and their effects in zebrafish embryos. (a) Schematic of the miRNA and the eGFP sensor vectors showing the miRNA target sites as well as the qRT-PCR primer locations. The miRNA vector contains a CMV promoter for ubiquitous expression in all tissues, and a DsRed Express reporter gene and the pre-miRNAs. The pre-miRNAs can be single (only one miRNA) or chained (containing more than one miRNA under the same promoter). The eGPF sensor miRNA also contains a CMV promoter, an eGFP reporter gene, and the mature miRNA target at the 3′ UTR of the eGFP gene. The miRNA target sites for zerg-2 are located in exons 9 (miRNA 1), 14 no. 1 (miRNA 2), 14 no. 2 (miRNA 3), and 3′ UTR (miRNA 4). The miRNA targets for zerg-3 are located in exon 8 (miRNA 1), exon 15 (miRNA 2), and two targets located on the 3′ UTR. (b) The injection of eGFP sensor mRNA alone produces embryos that exhibit green fluorescence only, and embryos injected with both eGFP sensor and miRNA mRNA exhibit both green and red fluorescence. (c) An example of embryos injected with either miRNA with eGFP sensor or scrambled miRNA (negative control) with eGFP sensor. The ∗ indicates the presence of the midbrain hindbrain boundary.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303736&req=5

fig1: miRNA constructs and their effects in zebrafish embryos. (a) Schematic of the miRNA and the eGFP sensor vectors showing the miRNA target sites as well as the qRT-PCR primer locations. The miRNA vector contains a CMV promoter for ubiquitous expression in all tissues, and a DsRed Express reporter gene and the pre-miRNAs. The pre-miRNAs can be single (only one miRNA) or chained (containing more than one miRNA under the same promoter). The eGPF sensor miRNA also contains a CMV promoter, an eGFP reporter gene, and the mature miRNA target at the 3′ UTR of the eGFP gene. The miRNA target sites for zerg-2 are located in exons 9 (miRNA 1), 14 no. 1 (miRNA 2), 14 no. 2 (miRNA 3), and 3′ UTR (miRNA 4). The miRNA targets for zerg-3 are located in exon 8 (miRNA 1), exon 15 (miRNA 2), and two targets located on the 3′ UTR. (b) The injection of eGFP sensor mRNA alone produces embryos that exhibit green fluorescence only, and embryos injected with both eGFP sensor and miRNA mRNA exhibit both green and red fluorescence. (c) An example of embryos injected with either miRNA with eGFP sensor or scrambled miRNA (negative control) with eGFP sensor. The ∗ indicates the presence of the midbrain hindbrain boundary.
Mentions: As the pCS2+ vector does not contain an eGFP reporter gene, one had to be inserted into the vector before the eGFP sensor vector could be used. The Kozak sequence and the eGFP coding region was excised from pT2AL200R150G (a gift from Dr Koichi Kawakami, National Institute of Genetics) using BamHI and ClaI restriction enzymes. The pCS2+ vector was restriction enzyme digested with BamHI and ClaI and the eGFP coding region was cloned into the linearised vector (Figure 1).

Bottom Line: The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans.The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest.In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT
The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

Show MeSH
Related in: MedlinePlus