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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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Related in: MedlinePlus

In vivo eGFP sensor assay for zerg-3 miRNAs. (a) zerg-3 miRNA 1 with eGFP knockdown at approximately 90%. (b) zerg-3 miRNA 2 with eGFP knockdown at approximately 45% (the results are not statically significant). (c) zerg-3 miRNA 3 with eGFP knockdown at approximately 98%. (d) zer-g3 miRNA 4 with eGFP knockdown at approximately 77%. **P < 0.01; ***P < 0.001. All statistical analysis was carried out using one-way ANOVA. The percentage of fluorescence emitted by the injected embryos was determined by comparison to negative control embryos (those injected with scrambled miRNA).
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fig3: In vivo eGFP sensor assay for zerg-3 miRNAs. (a) zerg-3 miRNA 1 with eGFP knockdown at approximately 90%. (b) zerg-3 miRNA 2 with eGFP knockdown at approximately 45% (the results are not statically significant). (c) zerg-3 miRNA 3 with eGFP knockdown at approximately 98%. (d) zer-g3 miRNA 4 with eGFP knockdown at approximately 77%. **P < 0.01; ***P < 0.001. All statistical analysis was carried out using one-way ANOVA. The percentage of fluorescence emitted by the injected embryos was determined by comparison to negative control embryos (those injected with scrambled miRNA).

Mentions: All eight miRNAs were able to decrease eGFP expression (Figures 2 and 3). Zerg-2 miRNA 4 caused the greatest level of eGFP expression knockdown (approximately 88%) with zerg-2 miRNA 1 and 2 causing approximately 69–73% eGFP knockdown (Figure 2). Zerg-2 miRNA 3 was only able to cause 32% knockdown (Figure 2). Zerg3 miRNA 3 caused the greatest level of eGFP knockdown (approximately 98%) with zerg3 miRNA 1 and 4 causing approximately 77–90% knockdown (Figure 3); the extent of knockdown caused by Zerg-3 miRNA 2 was not statistically significant. The results from the in vivo sensor assay demonstrate that the Block-iT miRNA backbone is functional in zebrafish and the custom-designed miRNA is capable of causing gene knockdown as assessed by the in vivo sensor system.


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

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

In vivo eGFP sensor assay for zerg-3 miRNAs. (a) zerg-3 miRNA 1 with eGFP knockdown at approximately 90%. (b) zerg-3 miRNA 2 with eGFP knockdown at approximately 45% (the results are not statically significant). (c) zerg-3 miRNA 3 with eGFP knockdown at approximately 98%. (d) zer-g3 miRNA 4 with eGFP knockdown at approximately 77%. **P < 0.01; ***P < 0.001. All statistical analysis was carried out using one-way ANOVA. The percentage of fluorescence emitted by the injected embryos was determined by comparison to negative control embryos (those injected with scrambled miRNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: In vivo eGFP sensor assay for zerg-3 miRNAs. (a) zerg-3 miRNA 1 with eGFP knockdown at approximately 90%. (b) zerg-3 miRNA 2 with eGFP knockdown at approximately 45% (the results are not statically significant). (c) zerg-3 miRNA 3 with eGFP knockdown at approximately 98%. (d) zer-g3 miRNA 4 with eGFP knockdown at approximately 77%. **P < 0.01; ***P < 0.001. All statistical analysis was carried out using one-way ANOVA. The percentage of fluorescence emitted by the injected embryos was determined by comparison to negative control embryos (those injected with scrambled miRNA).
Mentions: All eight miRNAs were able to decrease eGFP expression (Figures 2 and 3). Zerg-2 miRNA 4 caused the greatest level of eGFP expression knockdown (approximately 88%) with zerg-2 miRNA 1 and 2 causing approximately 69–73% eGFP knockdown (Figure 2). Zerg-2 miRNA 3 was only able to cause 32% knockdown (Figure 2). Zerg3 miRNA 3 caused the greatest level of eGFP knockdown (approximately 98%) with zerg3 miRNA 1 and 4 causing approximately 77–90% knockdown (Figure 3); the extent of knockdown caused by Zerg-3 miRNA 2 was not statistically significant. The results from the in vivo sensor assay demonstrate that the Block-iT miRNA backbone is functional in zebrafish and the custom-designed miRNA is capable of causing gene knockdown as assessed by the in vivo sensor system.

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