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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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Effect of feeder cells expressing zebrafish Lif, Fgf2 and Gdnf on FGSCs in 6-week cultures.Photomicrographs showing two DsRed-positive FGSC colonies containing 100 or more cells (black arrows) under (A) bright field and (B) merged with UV (DsRed). (C) RT-PCR analysis of RNA isolated from 6-week FGSC cultures showing expression of germ cell specific maker genes, dazl, dnd, nanos3, vasa and ziwi. RT: reverse transcription; Scale bar = 20 µm.
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pone-0062660-g005: Effect of feeder cells expressing zebrafish Lif, Fgf2 and Gdnf on FGSCs in 6-week cultures.Photomicrographs showing two DsRed-positive FGSC colonies containing 100 or more cells (black arrows) under (A) bright field and (B) merged with UV (DsRed). (C) RT-PCR analysis of RNA isolated from 6-week FGSC cultures showing expression of germ cell specific maker genes, dazl, dnd, nanos3, vasa and ziwi. RT: reverse transcription; Scale bar = 20 µm.

Mentions: To optimize the FGSC culture conditions, OFC3 was transfected with a plasmid encoding zebrafish Lif and stable colonies (OFC3L) that expressed the factor were isolated. Results showed that both FGSC colony formation and cell proliferation were significantly enhanced in the presence of OFC3L compared to cultures grown on the parent OFC3 feeder line and supplemented with recombinant mammalian LIF (Fig. 4A, B). To further optimize the FGSC culture conditions, the OFC3L feeder cells were engineered to stably express either zebrafish Gdnf (OFC3LG) or Fgf2 (OFC3LF) along with Lif and the mitogenic activity of each feeder line was tested. Although there was no significant effect of OFC3LG or OFC3LF feeder layers on the number of FGSC colonies that formed in culture (Fig. 4C), the presence of OFC3LF did enhance FGSC proliferation resulting in larger colonies (Fig. 4D) after 3 weeks of culture. The use of a feeder layer comprised of both OFC3LF and OFC3LG (1∶1 mix) resulted in optimal FGSC proliferation after 3 weeks in culture (Fig. 4D, E). After 6 weeks, the total number of FGSC colonies significantly decreased on each of the feeder layers when compared to the 3 week cultures (Table S1 and Fig. S1A); however, the remaining colonies were larger and total number of cells contained in each well increased. In the wells containing OFC3LF and OFC3LG feeder layers, the total number of FGSCs increased approximately 3-fold during this period (Fig. S1B). The largest colonies examined in the 6-week-old cultures contained up to 100 FGSCs (Fig. 5A,B) and the average number of FGSCs per colony increased 4-fold compared to the 3-week-old cultures (Fig. S1C). The results indicated that the average doubling time of the FGSCs on OFC3LF and OFC3LG feeder layers was approximately 10 days. RT-PCR analysis revealed that at 6-weeks the cultures continued to express germ cell-specific markers, dazl, dnd, nanos3, vasa and ziwi (Fig. 5C). In cultures maintained on OFC3 feeder cells and supplemented with recombinant mammalian LIF, FGF2 or GDNF, the number of FGSCs significantly decreased over 3 weeks and were nearly absent by 6 weeks (Fig. S1B) demonstrating that the recombinant mammalian growth factors were not able to sustain the FGSCs in culture.


Production of zebrafish offspring from cultured female germline stem cells.

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

Effect of feeder cells expressing zebrafish Lif, Fgf2 and Gdnf on FGSCs in 6-week cultures.Photomicrographs showing two DsRed-positive FGSC colonies containing 100 or more cells (black arrows) under (A) bright field and (B) merged with UV (DsRed). (C) RT-PCR analysis of RNA isolated from 6-week FGSC cultures showing expression of germ cell specific maker genes, dazl, dnd, nanos3, vasa and ziwi. RT: reverse transcription; Scale bar = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643964&req=5

pone-0062660-g005: Effect of feeder cells expressing zebrafish Lif, Fgf2 and Gdnf on FGSCs in 6-week cultures.Photomicrographs showing two DsRed-positive FGSC colonies containing 100 or more cells (black arrows) under (A) bright field and (B) merged with UV (DsRed). (C) RT-PCR analysis of RNA isolated from 6-week FGSC cultures showing expression of germ cell specific maker genes, dazl, dnd, nanos3, vasa and ziwi. RT: reverse transcription; Scale bar = 20 µm.
Mentions: To optimize the FGSC culture conditions, OFC3 was transfected with a plasmid encoding zebrafish Lif and stable colonies (OFC3L) that expressed the factor were isolated. Results showed that both FGSC colony formation and cell proliferation were significantly enhanced in the presence of OFC3L compared to cultures grown on the parent OFC3 feeder line and supplemented with recombinant mammalian LIF (Fig. 4A, B). To further optimize the FGSC culture conditions, the OFC3L feeder cells were engineered to stably express either zebrafish Gdnf (OFC3LG) or Fgf2 (OFC3LF) along with Lif and the mitogenic activity of each feeder line was tested. Although there was no significant effect of OFC3LG or OFC3LF feeder layers on the number of FGSC colonies that formed in culture (Fig. 4C), the presence of OFC3LF did enhance FGSC proliferation resulting in larger colonies (Fig. 4D) after 3 weeks of culture. The use of a feeder layer comprised of both OFC3LF and OFC3LG (1∶1 mix) resulted in optimal FGSC proliferation after 3 weeks in culture (Fig. 4D, E). After 6 weeks, the total number of FGSC colonies significantly decreased on each of the feeder layers when compared to the 3 week cultures (Table S1 and Fig. S1A); however, the remaining colonies were larger and total number of cells contained in each well increased. In the wells containing OFC3LF and OFC3LG feeder layers, the total number of FGSCs increased approximately 3-fold during this period (Fig. S1B). The largest colonies examined in the 6-week-old cultures contained up to 100 FGSCs (Fig. 5A,B) and the average number of FGSCs per colony increased 4-fold compared to the 3-week-old cultures (Fig. S1C). The results indicated that the average doubling time of the FGSCs on OFC3LF and OFC3LG feeder layers was approximately 10 days. RT-PCR analysis revealed that at 6-weeks the cultures continued to express germ cell-specific markers, dazl, dnd, nanos3, vasa and ziwi (Fig. 5C). In cultures maintained on OFC3 feeder cells and supplemented with recombinant mammalian LIF, FGF2 or GDNF, the number of FGSCs significantly decreased over 3 weeks and were nearly absent by 6 weeks (Fig. S1B) demonstrating that the recombinant mammalian growth factors were not able to sustain the FGSCs in culture.

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