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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

Kinetics of YFP induction in fry.Transgenic fries were treated with the indicated concentrations of Shield-1 or 0.1% ethanol (vehicle). After 48 hours, the water was exchanged and the imaging was continued up to 72 and 96 hours to monitor the reversibility of the induction. (A-C) Fluorescence images of a representative individual treated with 100 nM Shield-1; dorsal view, head to the right. The faint fluorescence seen in transgenic individuals after 1 hour (A) increases to maximum after 48 hours (B) and decreases to background level after 96 hours of the experiment (C). (D) Fluorescence values (area fluorescence, arbitrary units) after treatment of fry with the indicated concentrations of Shield-1. Data points of three individuals are shown for the vehicle control, 5–6 individuals for the other groups (mean with standard deviation). The images were taken over a period of 96hours with an exposure time of 10 seconds. The monochrome images in A-C were colored for clarity.
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pone.0131252.g001: Kinetics of YFP induction in fry.Transgenic fries were treated with the indicated concentrations of Shield-1 or 0.1% ethanol (vehicle). After 48 hours, the water was exchanged and the imaging was continued up to 72 and 96 hours to monitor the reversibility of the induction. (A-C) Fluorescence images of a representative individual treated with 100 nM Shield-1; dorsal view, head to the right. The faint fluorescence seen in transgenic individuals after 1 hour (A) increases to maximum after 48 hours (B) and decreases to background level after 96 hours of the experiment (C). (D) Fluorescence values (area fluorescence, arbitrary units) after treatment of fry with the indicated concentrations of Shield-1. Data points of three individuals are shown for the vehicle control, 5–6 individuals for the other groups (mean with standard deviation). The images were taken over a period of 96hours with an exposure time of 10 seconds. The monochrome images in A-C were colored for clarity.

Mentions: Ten freshly hatched fry (F4 generation) per concentration were treated with Shield-1. An induction of YFP could be observed within a few hours in transgenic fry (Fig 1). Each individual was genotyped after the experiment and the data of the transgenics were used for the analysis of leakiness and the kinetics of induction in vivo. The presence of autofluorescent yolk and leucophores defines the basal level of fluorescence observed in non-transgenic fish and transgenics, respectively (Fig 1C).


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)

Kinetics of YFP induction in fry.Transgenic fries were treated with the indicated concentrations of Shield-1 or 0.1% ethanol (vehicle). After 48 hours, the water was exchanged and the imaging was continued up to 72 and 96 hours to monitor the reversibility of the induction. (A-C) Fluorescence images of a representative individual treated with 100 nM Shield-1; dorsal view, head to the right. The faint fluorescence seen in transgenic individuals after 1 hour (A) increases to maximum after 48 hours (B) and decreases to background level after 96 hours of the experiment (C). (D) Fluorescence values (area fluorescence, arbitrary units) after treatment of fry with the indicated concentrations of Shield-1. Data points of three individuals are shown for the vehicle control, 5–6 individuals for the other groups (mean with standard deviation). The images were taken over a period of 96hours with an exposure time of 10 seconds. The monochrome images in A-C were colored for clarity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493054&req=5

pone.0131252.g001: Kinetics of YFP induction in fry.Transgenic fries were treated with the indicated concentrations of Shield-1 or 0.1% ethanol (vehicle). After 48 hours, the water was exchanged and the imaging was continued up to 72 and 96 hours to monitor the reversibility of the induction. (A-C) Fluorescence images of a representative individual treated with 100 nM Shield-1; dorsal view, head to the right. The faint fluorescence seen in transgenic individuals after 1 hour (A) increases to maximum after 48 hours (B) and decreases to background level after 96 hours of the experiment (C). (D) Fluorescence values (area fluorescence, arbitrary units) after treatment of fry with the indicated concentrations of Shield-1. Data points of three individuals are shown for the vehicle control, 5–6 individuals for the other groups (mean with standard deviation). The images were taken over a period of 96hours with an exposure time of 10 seconds. The monochrome images in A-C were colored for clarity.
Mentions: Ten freshly hatched fry (F4 generation) per concentration were treated with Shield-1. An induction of YFP could be observed within a few hours in transgenic fry (Fig 1). Each individual was genotyped after the experiment and the data of the transgenics were used for the analysis of leakiness and the kinetics of induction in vivo. The presence of autofluorescent yolk and leucophores defines the basal level of fluorescence observed in non-transgenic fish and transgenics, respectively (Fig 1C).

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