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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

Induced fluorescence of testis cells analyzed by flow cytometry.Primary testis cells of hemizygous Ola-Tg(DD-YFP)13 were analyzed for size (FSC), fluorescence (GFP) and DNA content (Gate R1; Hoechst staining, not shown). The quadrants Q1 and Q2 contain cells with a DNA content of 2C to 4C, i.e. somatic cells and germ cells prior to meiosis. The plots show the data of 129–154,000 single cells. (A) The fluorescence in a sample of a non-transgenic FLFII and was plotted against the cell size. (B) In the non-induced control virtually no additional fluorescence is observed in Q2 compared to FLFII. (C) Treatment with 1 μM Shield-1 causes a strong induction of fluorescence in the largest cells that comprise the stem cell fraction. Absolute fluorescence and number of cells in Q2 are increased. (D) For comparison, the GFP-positive stem cells in FLF-Tg(oct4-EGFP)18 are shown [17]. In this line, the fluorescence decreases during diffentiation (arrow). In contrast, a cell fraction of similar size has an elevated fluorescence after induction (arrow in C). These cells represent the mitotically active type B spermatogonia that actively transcribe the actb-driven DD-YFP but not the oct4-driven EGFP.
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pone.0131252.g004: Induced fluorescence of testis cells analyzed by flow cytometry.Primary testis cells of hemizygous Ola-Tg(DD-YFP)13 were analyzed for size (FSC), fluorescence (GFP) and DNA content (Gate R1; Hoechst staining, not shown). The quadrants Q1 and Q2 contain cells with a DNA content of 2C to 4C, i.e. somatic cells and germ cells prior to meiosis. The plots show the data of 129–154,000 single cells. (A) The fluorescence in a sample of a non-transgenic FLFII and was plotted against the cell size. (B) In the non-induced control virtually no additional fluorescence is observed in Q2 compared to FLFII. (C) Treatment with 1 μM Shield-1 causes a strong induction of fluorescence in the largest cells that comprise the stem cell fraction. Absolute fluorescence and number of cells in Q2 are increased. (D) For comparison, the GFP-positive stem cells in FLF-Tg(oct4-EGFP)18 are shown [17]. In this line, the fluorescence decreases during diffentiation (arrow). In contrast, a cell fraction of similar size has an elevated fluorescence after induction (arrow in C). These cells represent the mitotically active type B spermatogonia that actively transcribe the actb-driven DD-YFP but not the oct4-driven EGFP.

Mentions: The gonadal stem cells of the medaka are located in the outer tubular compartment of the testis and in the cradles of the ovary [23, 24]. Remarkably, these regions showed an inducible YFP fluorescence after in vivo treatment (Fig 3C and S3 Fig). For the testis, we quantified the induced fluorescence by flow cytometry (Fig 4). Primary testis cells were isolated from transgenic fish after 24 hours in vivo treatment. For comparison, cells of non-transgenic FLFII (Fig 4A) and cells of an oct4-EGFP reporter line that labels the stem cell fraction of the testis (Fig 4D) were isolated. Identical gates and filters were used for the analysis; the quadrants indicate the background fluorescence (Q1, Q3) on the horizonzal axis and the cells size of the somatic cells and pre-meiotic germ cells (Q1, Q2).


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)

Induced fluorescence of testis cells analyzed by flow cytometry.Primary testis cells of hemizygous Ola-Tg(DD-YFP)13 were analyzed for size (FSC), fluorescence (GFP) and DNA content (Gate R1; Hoechst staining, not shown). The quadrants Q1 and Q2 contain cells with a DNA content of 2C to 4C, i.e. somatic cells and germ cells prior to meiosis. The plots show the data of 129–154,000 single cells. (A) The fluorescence in a sample of a non-transgenic FLFII and was plotted against the cell size. (B) In the non-induced control virtually no additional fluorescence is observed in Q2 compared to FLFII. (C) Treatment with 1 μM Shield-1 causes a strong induction of fluorescence in the largest cells that comprise the stem cell fraction. Absolute fluorescence and number of cells in Q2 are increased. (D) For comparison, the GFP-positive stem cells in FLF-Tg(oct4-EGFP)18 are shown [17]. In this line, the fluorescence decreases during diffentiation (arrow). In contrast, a cell fraction of similar size has an elevated fluorescence after induction (arrow in C). These cells represent the mitotically active type B spermatogonia that actively transcribe the actb-driven DD-YFP but not the oct4-driven EGFP.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131252.g004: Induced fluorescence of testis cells analyzed by flow cytometry.Primary testis cells of hemizygous Ola-Tg(DD-YFP)13 were analyzed for size (FSC), fluorescence (GFP) and DNA content (Gate R1; Hoechst staining, not shown). The quadrants Q1 and Q2 contain cells with a DNA content of 2C to 4C, i.e. somatic cells and germ cells prior to meiosis. The plots show the data of 129–154,000 single cells. (A) The fluorescence in a sample of a non-transgenic FLFII and was plotted against the cell size. (B) In the non-induced control virtually no additional fluorescence is observed in Q2 compared to FLFII. (C) Treatment with 1 μM Shield-1 causes a strong induction of fluorescence in the largest cells that comprise the stem cell fraction. Absolute fluorescence and number of cells in Q2 are increased. (D) For comparison, the GFP-positive stem cells in FLF-Tg(oct4-EGFP)18 are shown [17]. In this line, the fluorescence decreases during diffentiation (arrow). In contrast, a cell fraction of similar size has an elevated fluorescence after induction (arrow in C). These cells represent the mitotically active type B spermatogonia that actively transcribe the actb-driven DD-YFP but not the oct4-driven EGFP.
Mentions: The gonadal stem cells of the medaka are located in the outer tubular compartment of the testis and in the cradles of the ovary [23, 24]. Remarkably, these regions showed an inducible YFP fluorescence after in vivo treatment (Fig 3C and S3 Fig). For the testis, we quantified the induced fluorescence by flow cytometry (Fig 4). Primary testis cells were isolated from transgenic fish after 24 hours in vivo treatment. For comparison, cells of non-transgenic FLFII (Fig 4A) and cells of an oct4-EGFP reporter line that labels the stem cell fraction of the testis (Fig 4D) were isolated. Identical gates and filters were used for the analysis; the quadrants indicate the background fluorescence (Q1, Q3) on the horizonzal axis and the cells size of the somatic cells and pre-meiotic germ cells (Q1, Q2).

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