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Production of zebrafish offspring from cultured female germline stem cells.

Wong TT, Tesfamichael A, Collodi P - PLoS ONE (2013)

Bottom Line: Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes.Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months.The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Purdue University, West Lafayette, Indiana, USA. Wong20@purdue.edu

ABSTRACT
Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.

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Production of Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish.(A,B) Diagram of the constructs used to produce the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish. A 4.8 kb fragment of the ziwi promoter [13] was used to drive expression of (A) neo and (B) DsRed. (C) Neo (green) and (D) Vasa (red) are expressed in the same ovarian germ cells of Tg(ziwi:neo). Higher magnification showing expression of (E) Neo (green) and (F) Vasa (red) in the oogonia (white arrows) of Tg(ziwi:neo); (G) merged photo of E and F. (H) DsRed was detected in the ovarian germ cells of Tg(ziwi:DsRed). Scale bar = 50 µm for C, D, H and 20 µm for E, F, G.
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pone-0062660-g001: Production of Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish.(A,B) Diagram of the constructs used to produce the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish. A 4.8 kb fragment of the ziwi promoter [13] was used to drive expression of (A) neo and (B) DsRed. (C) Neo (green) and (D) Vasa (red) are expressed in the same ovarian germ cells of Tg(ziwi:neo). Higher magnification showing expression of (E) Neo (green) and (F) Vasa (red) in the oogonia (white arrows) of Tg(ziwi:neo); (G) merged photo of E and F. (H) DsRed was detected in the ovarian germ cells of Tg(ziwi:DsRed). Scale bar = 50 µm for C, D, H and 20 µm for E, F, G.

Mentions: To establish FGSC cultures, we produced Tg(ziwi:neo);Tg(ziwi:DsRed) double transgenic zebrafish in which the FGSCs express Neo and DsRed. A 4.8-kb fragment of the ziwi promoter, previously shown to direct EGFP expression in ovarian germ cells including oogonial stem cells [13], was used (Fig. 1A, B) to generate the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic line of fish. Immunocytochemical analysis of ovarian tissue dissected from the transgenic fish confirmed that Neo is expressed in the Vasa-positive ovarian germ cells including oogonia (Fig. 1C–G). Fluorescence microscopy revealed the DsRed is also expressed in ovarian germ cells (Fig. 1H) of Tg(ziwi:DsRed).


Production of zebrafish offspring from cultured female germline stem cells.

Wong TT, Tesfamichael A, Collodi P - PLoS ONE (2013)

Production of Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish.(A,B) Diagram of the constructs used to produce the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish. A 4.8 kb fragment of the ziwi promoter [13] was used to drive expression of (A) neo and (B) DsRed. (C) Neo (green) and (D) Vasa (red) are expressed in the same ovarian germ cells of Tg(ziwi:neo). Higher magnification showing expression of (E) Neo (green) and (F) Vasa (red) in the oogonia (white arrows) of Tg(ziwi:neo); (G) merged photo of E and F. (H) DsRed was detected in the ovarian germ cells of Tg(ziwi:DsRed). Scale bar = 50 µm for C, D, H and 20 µm for E, F, G.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643964&req=5

pone-0062660-g001: Production of Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish.(A,B) Diagram of the constructs used to produce the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic fish. A 4.8 kb fragment of the ziwi promoter [13] was used to drive expression of (A) neo and (B) DsRed. (C) Neo (green) and (D) Vasa (red) are expressed in the same ovarian germ cells of Tg(ziwi:neo). Higher magnification showing expression of (E) Neo (green) and (F) Vasa (red) in the oogonia (white arrows) of Tg(ziwi:neo); (G) merged photo of E and F. (H) DsRed was detected in the ovarian germ cells of Tg(ziwi:DsRed). Scale bar = 50 µm for C, D, H and 20 µm for E, F, G.
Mentions: To establish FGSC cultures, we produced Tg(ziwi:neo);Tg(ziwi:DsRed) double transgenic zebrafish in which the FGSCs express Neo and DsRed. A 4.8-kb fragment of the ziwi promoter, previously shown to direct EGFP expression in ovarian germ cells including oogonial stem cells [13], was used (Fig. 1A, B) to generate the Tg(ziwi:neo) and Tg(ziwi:DsRed) transgenic line of fish. Immunocytochemical analysis of ovarian tissue dissected from the transgenic fish confirmed that Neo is expressed in the Vasa-positive ovarian germ cells including oogonia (Fig. 1C–G). Fluorescence microscopy revealed the DsRed is also expressed in ovarian germ cells (Fig. 1H) of Tg(ziwi:DsRed).

Bottom Line: Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes.Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months.The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Purdue University, West Lafayette, Indiana, USA. Wong20@purdue.edu

ABSTRACT
Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.

Show MeSH
Related in: MedlinePlus