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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 ovarian-somatic feeder cell (OFC) lines.(A) Diagram of the construct used to produce the Tg(gsdf:neo) fish. The plasmid contained a 3.8 kb gsdf promoter controlling expression of neo. (B) Neo (green) expressed in ovarian somatic cells, particularly the granulosa cells, of Tg(gsdf:neo). (C) The merger of (B) with the corresponding bright field photo. Photomicrograph showing 12 month-old (D) OFC1, (E) OFC3, (F) OFC5 and (G) OFC6 ovarian feeder cell cultures. Scale bar = 50 µm.
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pone-0062660-g002: Production of ovarian-somatic feeder cell (OFC) lines.(A) Diagram of the construct used to produce the Tg(gsdf:neo) fish. The plasmid contained a 3.8 kb gsdf promoter controlling expression of neo. (B) Neo (green) expressed in ovarian somatic cells, particularly the granulosa cells, of Tg(gsdf:neo). (C) The merger of (B) with the corresponding bright field photo. Photomicrograph showing 12 month-old (D) OFC1, (E) OFC3, (F) OFC5 and (G) OFC6 ovarian feeder cell cultures. Scale bar = 50 µm.

Mentions: Ovarian tissue dissected from 10- to 12-week old Tg(ziwi:neo);Tg(ziwi:DsRed) zebrafish was dissociated and partially purified through a Percoll gradient to obtain a cell fraction enriched for DsRed-positive ovarian germ cells [5]. Primary cell cultures were initiated from the cell fraction and ziwi-neo-expressing FGSCs were selected in G418. To propagate the FGSCs in culture, the cells were maintained on a growth-arrested feeder layer comprised of an ovarian-somatic feeder cell (OFC) line that was derived from Tg(gsdf:neo) transgenic zebrafish. The Tg(gsdf:neo) fish were generated using a 3.8-kb fragment of the gsdf promoter to drive neo expression (Fig. 2A) specifically in ovarian somatic cells of the transgenic fish (Fig. 2B, C). The gsdf-neo-expressing cell lines were obtained by G418 selection of ovarian primary cultures that were initiated from the Tg(gsdf:neo) fish. Four G418-resistant colonies (OFC1,3,5,6) were expanded to individual cell lines (Fig. 2D–G) and evaluated for use as feeder layers in FGSC cultures. DsRed-positive FGSCs, obtained by Percoll fractionation of Tg(ziwi:neo);Tg(ziwi:DsRed) ovarian tissue, were plated at clonal density onto a confluent layer of each feeder cell line and the growth of the FGSC clones were evaluated after 3 weeks. The results showed that a feeder layer consisting of either OFC1 or OFC3 promoted the proliferation of the FGSCs resulting in colonies of 4 or more G418-resistant and DsRed-positive cells (Fig. 3A–D). The morphology of the FGSCs in culture resembled the mitotically active oogonia found in the ovary [5] possessing a diameter of approximately 10 µm or less and a high nucleus-to-cytoplasm ratio with one to two nucleoli (Fig. 3A, B). Even after more than 3 weeks in culture on the ovarian cell feeder layer the FGSC colonies continued to express ziwi-DsRed (Fig. 3C). Although a few larger DsRed-positive ovarian germ cells resembling early-stage oocytes were initially observed in the cultures, these larger cells did not attach tightly to the feeder layer or proliferate in culture and were not present after 3 weeks. Most of the DsRed-positive cells were located within colonies and possessed an oogonium-like morphology. Since the results showed that OFC3 promoted FGSC colony formation and growth (Fig. 3E,F), this cell line was used for subsequent experiments.


Production of zebrafish offspring from cultured female germline stem cells.

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

Production of ovarian-somatic feeder cell (OFC) lines.(A) Diagram of the construct used to produce the Tg(gsdf:neo) fish. The plasmid contained a 3.8 kb gsdf promoter controlling expression of neo. (B) Neo (green) expressed in ovarian somatic cells, particularly the granulosa cells, of Tg(gsdf:neo). (C) The merger of (B) with the corresponding bright field photo. Photomicrograph showing 12 month-old (D) OFC1, (E) OFC3, (F) OFC5 and (G) OFC6 ovarian feeder cell cultures. Scale bar = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643964&req=5

pone-0062660-g002: Production of ovarian-somatic feeder cell (OFC) lines.(A) Diagram of the construct used to produce the Tg(gsdf:neo) fish. The plasmid contained a 3.8 kb gsdf promoter controlling expression of neo. (B) Neo (green) expressed in ovarian somatic cells, particularly the granulosa cells, of Tg(gsdf:neo). (C) The merger of (B) with the corresponding bright field photo. Photomicrograph showing 12 month-old (D) OFC1, (E) OFC3, (F) OFC5 and (G) OFC6 ovarian feeder cell cultures. Scale bar = 50 µm.
Mentions: Ovarian tissue dissected from 10- to 12-week old Tg(ziwi:neo);Tg(ziwi:DsRed) zebrafish was dissociated and partially purified through a Percoll gradient to obtain a cell fraction enriched for DsRed-positive ovarian germ cells [5]. Primary cell cultures were initiated from the cell fraction and ziwi-neo-expressing FGSCs were selected in G418. To propagate the FGSCs in culture, the cells were maintained on a growth-arrested feeder layer comprised of an ovarian-somatic feeder cell (OFC) line that was derived from Tg(gsdf:neo) transgenic zebrafish. The Tg(gsdf:neo) fish were generated using a 3.8-kb fragment of the gsdf promoter to drive neo expression (Fig. 2A) specifically in ovarian somatic cells of the transgenic fish (Fig. 2B, C). The gsdf-neo-expressing cell lines were obtained by G418 selection of ovarian primary cultures that were initiated from the Tg(gsdf:neo) fish. Four G418-resistant colonies (OFC1,3,5,6) were expanded to individual cell lines (Fig. 2D–G) and evaluated for use as feeder layers in FGSC cultures. DsRed-positive FGSCs, obtained by Percoll fractionation of Tg(ziwi:neo);Tg(ziwi:DsRed) ovarian tissue, were plated at clonal density onto a confluent layer of each feeder cell line and the growth of the FGSC clones were evaluated after 3 weeks. The results showed that a feeder layer consisting of either OFC1 or OFC3 promoted the proliferation of the FGSCs resulting in colonies of 4 or more G418-resistant and DsRed-positive cells (Fig. 3A–D). The morphology of the FGSCs in culture resembled the mitotically active oogonia found in the ovary [5] possessing a diameter of approximately 10 µm or less and a high nucleus-to-cytoplasm ratio with one to two nucleoli (Fig. 3A, B). Even after more than 3 weeks in culture on the ovarian cell feeder layer the FGSC colonies continued to express ziwi-DsRed (Fig. 3C). Although a few larger DsRed-positive ovarian germ cells resembling early-stage oocytes were initially observed in the cultures, these larger cells did not attach tightly to the feeder layer or proliferate in culture and were not present after 3 weeks. Most of the DsRed-positive cells were located within colonies and possessed an oogonium-like morphology. Since the results showed that OFC3 promoted FGSC colony formation and growth (Fig. 3E,F), this cell line was used for subsequent experiments.

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