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Derivation and characterization of haploid embryonic stem cell cultures in medaka fish

Nature Protocols volume 5, pages 1418–1430 (2010)Cite this article

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Abstract

Embryonic stem (ES) cells are pluripotent cells capable of differentiation into various cell types. Haploid ES cells elegantly combine the advantages of haploidy and pluripotency and offer a unique in vitro system for genetic analyses of molecular, cellular and developmental events in various cell lineages. Our recent success in generating haploid ES cell lines from gynogenetic embryos of the medaka fish suggests that haploidy can support ES cell derivation and maintenance in a vertebrate. In this study, we present a step-by-step protocol for derivation and characterization of medaka haploid ES cells. We have used this procedure to produce three haploid ES cell lines from five primary cultures. It takes about 15 weeks to generate stable cultures, 5–8 weeks to obtain pure haploid cells and 5–6 weeks to characterize ES cells in vitro and in vivo.

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Figure 1
Figure 2: Injection needle and equipment setup for cell transplantation system.
Figure 3: Pluripotency of haploid ES cells.
Figure 4: Derivation of medaka haploid ES cell cultures.

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Acknowledgements

We thank J. Deng for fish breeding. This work was supported by the Biomedical Research Council of Singapore (R-08-1-21-19-585 and SBIC-SSCC C-002-2007) and by the Lee Hiok Kwee donation from the National University of Singapore (R-154-000-153-720).

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Authors and Affiliations

  1. Department of Biological Sciences, National University of Singapore, Singapore

    Meisheng Yi, Ni Hong & Yunhan Hong

  2. Faculty of Life Sciences, Hubei University, Wuhan, Hubei, China

    Meisheng Yi

  3. Yangtze Fisheries Institute, Chinese Academy of Aquatic Sciences, Jingzhou, Hubei, China

    Yunhan Hong

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  1. Meisheng Yi
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Contributions

M.Y. designed and carried out experiments, analyzed data and wrote the paper. N.H. carried out experiments. Y.H. supervised the project, designed experiments and wrote and approved the final paper.

Corresponding author

Correspondence to Yunhan Hong.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Genes and primers used for RT-PCR analyses (PDF 47 kb)

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Yi, M., Hong, N. & Hong, Y. Derivation and characterization of haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5, 1418–1430 (2010). https://doi.org/10.1038/nprot.2010.104

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