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Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka.

Froschauer A, Kube L, Kegler A, Rieger C, Gutzeit HO - PLoS ONE (2015)

Bottom Line: Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease.We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life.Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, Technische Universität Dresden, Dresden, Germany.

ABSTRACT
Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

No MeSH data available.


Related in: MedlinePlus

Background fluorescence in transgenic fry of Ola-Tg(actin-DD-YFP)13.The fluorescence of hemizygous transgenic individuals treated with 0.1% ethanol and non-transgenic siblings was analyzed. (A) non-transgenic fry, (B) transgenic, non-induced fry. Monochrome images were colored for clarity, head to the right. (C) Box plot (25–75%), average (open square) and mean (line) of the fluorescence (arbitrary units) are shown. The values are normally distributed (Kolmogorow-Smirnov-test) in all samples and are significantly higher (asterisks, p≤0.05, ANOVA) for transgenics compared to non-transgenic fish within the first 72 hours of the experiment.
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pone.0131252.g002: Background fluorescence in transgenic fry of Ola-Tg(actin-DD-YFP)13.The fluorescence of hemizygous transgenic individuals treated with 0.1% ethanol and non-transgenic siblings was analyzed. (A) non-transgenic fry, (B) transgenic, non-induced fry. Monochrome images were colored for clarity, head to the right. (C) Box plot (25–75%), average (open square) and mean (line) of the fluorescence (arbitrary units) are shown. The values are normally distributed (Kolmogorow-Smirnov-test) in all samples and are significantly higher (asterisks, p≤0.05, ANOVA) for transgenics compared to non-transgenic fish within the first 72 hours of the experiment.

Mentions: Non-induced individuals of the line Ola-Tg(actin-DD-YFP)13 (Fig 1D) showed a weak fluorescence that is merely detectable by eye in the tail and somites (Fig 2A & 2B) but could be quantified by image analysis (Fig 2C & S2 Fig). The transgenic individuals show twice the area fluorescence (arbitrary units) than non-transgenics at the beginning of the analysis. Two factors contribute to the observed decrease of fluorescence. First, the yolk mass is autofluorescent and is metabolically degraded over time in both groups. Second, the yolk is enclosed by epithelia of the gut and epidermis. These epithelia are fluorescent in transgenic fry, hence leading to a disproportional contribution to the total area fluorescence within the first 48 hours (Fig 2C). At the end of the experiment the fluorescence values of the two groups are converging.


Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka.

Froschauer A, Kube L, Kegler A, Rieger C, Gutzeit HO - PLoS ONE (2015)

Background fluorescence in transgenic fry of Ola-Tg(actin-DD-YFP)13.The fluorescence of hemizygous transgenic individuals treated with 0.1% ethanol and non-transgenic siblings was analyzed. (A) non-transgenic fry, (B) transgenic, non-induced fry. Monochrome images were colored for clarity, head to the right. (C) Box plot (25–75%), average (open square) and mean (line) of the fluorescence (arbitrary units) are shown. The values are normally distributed (Kolmogorow-Smirnov-test) in all samples and are significantly higher (asterisks, p≤0.05, ANOVA) for transgenics compared to non-transgenic fish within the first 72 hours of the experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493054&req=5

pone.0131252.g002: Background fluorescence in transgenic fry of Ola-Tg(actin-DD-YFP)13.The fluorescence of hemizygous transgenic individuals treated with 0.1% ethanol and non-transgenic siblings was analyzed. (A) non-transgenic fry, (B) transgenic, non-induced fry. Monochrome images were colored for clarity, head to the right. (C) Box plot (25–75%), average (open square) and mean (line) of the fluorescence (arbitrary units) are shown. The values are normally distributed (Kolmogorow-Smirnov-test) in all samples and are significantly higher (asterisks, p≤0.05, ANOVA) for transgenics compared to non-transgenic fish within the first 72 hours of the experiment.
Mentions: Non-induced individuals of the line Ola-Tg(actin-DD-YFP)13 (Fig 1D) showed a weak fluorescence that is merely detectable by eye in the tail and somites (Fig 2A & 2B) but could be quantified by image analysis (Fig 2C & S2 Fig). The transgenic individuals show twice the area fluorescence (arbitrary units) than non-transgenics at the beginning of the analysis. Two factors contribute to the observed decrease of fluorescence. First, the yolk mass is autofluorescent and is metabolically degraded over time in both groups. Second, the yolk is enclosed by epithelia of the gut and epidermis. These epithelia are fluorescent in transgenic fry, hence leading to a disproportional contribution to the total area fluorescence within the first 48 hours (Fig 2C). At the end of the experiment the fluorescence values of the two groups are converging.

Bottom Line: Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease.We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life.Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, Technische Universität Dresden, Dresden, Germany.

ABSTRACT
Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

No MeSH data available.


Related in: MedlinePlus