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In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura.

Langer CC, Ejsmont RK, Schönbauer C, Schnorrer F, Tomancak P - PLoS ONE (2010)

Bottom Line: We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles.Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Biochemistry, Martinsried, Germany.

ABSTRACT

Background: Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/principal findings: We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/significance: The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

Show MeSH
Phenotypic rescue of sar1 by D. pseudoobscura fosmid.(a–d) Fading Z- and M-line or loss of sarcomeres in Mef2-GAL4/UAS-sar1-IR (TF34191) (b, c) is rescued by FlyFos-pse-sar1(d) to wild type (a). Z-lines are visualised with anti-Kettin (red), M-lines with anti-Mhc antibody (green); size bar corresponds to 50 µm. (e) Quantification of larval length in Mef2-GAL4/UAS-sar1-IR larvae (red), compared to FlyFos-pse-sar1 rescued (blue) and wild type (green). Larvae 72–96 h after egg laying were assayed. Error bars indicate standard error of the mean (SEM), ***p<0.0001 (unpaired two-tailed t-test) compared to rescued larvae.
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pone-0008928-g005: Phenotypic rescue of sar1 by D. pseudoobscura fosmid.(a–d) Fading Z- and M-line or loss of sarcomeres in Mef2-GAL4/UAS-sar1-IR (TF34191) (b, c) is rescued by FlyFos-pse-sar1(d) to wild type (a). Z-lines are visualised with anti-Kettin (red), M-lines with anti-Mhc antibody (green); size bar corresponds to 50 µm. (e) Quantification of larval length in Mef2-GAL4/UAS-sar1-IR larvae (red), compared to FlyFos-pse-sar1 rescued (blue) and wild type (green). Larvae 72–96 h after egg laying were assayed. Error bars indicate standard error of the mean (SEM), ***p<0.0001 (unpaired two-tailed t-test) compared to rescued larvae.

Mentions: Finally we investigated the small GTPase sar1 implicated in vesicle transport [41] and heart formation in the embryo [42]. Knock-down of sar1 in muscle causes a muscle sarcomere phenotype. Both the myosin thick filaments and the Z-line anchoring the actin filaments show a “fading-Z” phenotype or in extreme cases we observe a partial loss of sarcomeres (Figure 5a–c). The FlyFos-pse-sar1 completely rescues this sarcomere phenotype (Figure 5d) demonstrating a specific role of sar1 for sarcomere formation and in turn larval growth (Figure 5e).


In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura.

Langer CC, Ejsmont RK, Schönbauer C, Schnorrer F, Tomancak P - PLoS ONE (2010)

Phenotypic rescue of sar1 by D. pseudoobscura fosmid.(a–d) Fading Z- and M-line or loss of sarcomeres in Mef2-GAL4/UAS-sar1-IR (TF34191) (b, c) is rescued by FlyFos-pse-sar1(d) to wild type (a). Z-lines are visualised with anti-Kettin (red), M-lines with anti-Mhc antibody (green); size bar corresponds to 50 µm. (e) Quantification of larval length in Mef2-GAL4/UAS-sar1-IR larvae (red), compared to FlyFos-pse-sar1 rescued (blue) and wild type (green). Larvae 72–96 h after egg laying were assayed. Error bars indicate standard error of the mean (SEM), ***p<0.0001 (unpaired two-tailed t-test) compared to rescued larvae.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2812509&req=5

pone-0008928-g005: Phenotypic rescue of sar1 by D. pseudoobscura fosmid.(a–d) Fading Z- and M-line or loss of sarcomeres in Mef2-GAL4/UAS-sar1-IR (TF34191) (b, c) is rescued by FlyFos-pse-sar1(d) to wild type (a). Z-lines are visualised with anti-Kettin (red), M-lines with anti-Mhc antibody (green); size bar corresponds to 50 µm. (e) Quantification of larval length in Mef2-GAL4/UAS-sar1-IR larvae (red), compared to FlyFos-pse-sar1 rescued (blue) and wild type (green). Larvae 72–96 h after egg laying were assayed. Error bars indicate standard error of the mean (SEM), ***p<0.0001 (unpaired two-tailed t-test) compared to rescued larvae.
Mentions: Finally we investigated the small GTPase sar1 implicated in vesicle transport [41] and heart formation in the embryo [42]. Knock-down of sar1 in muscle causes a muscle sarcomere phenotype. Both the myosin thick filaments and the Z-line anchoring the actin filaments show a “fading-Z” phenotype or in extreme cases we observe a partial loss of sarcomeres (Figure 5a–c). The FlyFos-pse-sar1 completely rescues this sarcomere phenotype (Figure 5d) demonstrating a specific role of sar1 for sarcomere formation and in turn larval growth (Figure 5e).

Bottom Line: We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles.Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Biochemistry, Martinsried, Germany.

ABSTRACT

Background: Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/principal findings: We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/significance: The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

Show MeSH