Abstract
The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.
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References
Okutsu T, Yano A, Nagasawa K, Shikina S, Kobayashi T, Takeuchi Y et al (2006) Manipulation of fish germ cell: visualization, cryopreservation and transplantation. J Reprod Dev 52:685–693
Robles V, Riesco MF, Psenicka M, Saito T, Valcarce DG, Cabrita E et al (2017) Biology of teleost primordial germ cells (PGCs) and spermatogonia: biotechnological applications. Aquaculture 472:4–20. https://doi.org/10.1016/j.aquaculture.2016.03.004
Yamaha E, Saito T, Goto R, Arai K (2007) Developmental biotechnology for aquaculture, with special reference to surrogate production in teleost fishes. J Sea Res 58:8–22. https://doi.org/10.1016/j.seares.2007.02.003
Kawakami Y, Goto R, Saito T, Fujimoto T, Higaki S, Takahashi Y et al (2010) Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids. Int J Dev Biol 54:1493–1501. https://doi.org/10.1387/ijdb.093059yk
Tzung K-W, Goto R, Saju JM, Sreenivasan R, Saito T, Arai K et al (2014) Early depletion of primordial germ cells in zebrafish promotes testis formation. Stem Cell Reports 4:61–73. https://doi.org/10.1016/j.stemcr.2014.10.011
Raz E (2003) Primordial germ-cell development: the zebrafish perspective. Nat Rev Genet 4:690
Saito T, Psenicka M, Goto R, Adachi S, Inoue K, Arai K et al (2014) The origin and migration of primordial germ cells in sturgeons. PLoS One 9:e86861. https://doi.org/10.1371/journal.pone.0086861
Saito T, Fujimoto T, Maegawa S, Inoue K, Tanaka M, Arai K et al (2006) Visualization of primordial germ cells in vivo using GFP-nos1 3′UTR mRNA. Int J Dev Biol 50:691–699
Benfey TJ, Sutterlin AM (1984) Triploidy induced by heat shock and hydrostatic pressure in landlocked Atlantic salmon (Salmo salar L.). Aquaculture 36:359–367
Saito T, Güralp H, Iegorova V, Rodina M, Psenicka M (2018) Elimination of primordial germ cells in sturgeon embryos by ultraviolet irradiation. Biol Reprod 99(3):556–564
Weidinger G, Slanchev JK, Dumstrei K, Wise C, Lovell-Badge R, Thisse C et al (2003) Dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13:1429–1434
Fujimoto T, Nishimura T, Goto-Kazeto R, Kawakami Y, Yamaha E, Arai K (2010) Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish. Proc Natl Acad Sci 107:17211–17216
Goto R, Saito T, Takeda T, Fujimoto T, Takagi M, Arai K et al (2012) Germ cells are not the primary factor for sexual fate determination in goldfish. Dev Biol 370:98–109
Hong N, Li M, Yuan Y, Wang T, Yi M, Xu H et al (2016) Dnd is a critical specifier of primordial germ cells in the medaka fish. Stem Cell Reports 6:411–421
Saito T, Goto-Kazeto R, Arai K, Yamaha E (2008) Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation. Biol Reprod 78:159–166
Raz E, Reichman-Fried M (2006) Attraction rules: germ cell migration in zebrafish. Curr Opin Genet Dev 16:355–359
Doitsidou M, Reichman-Fried M, Stebler J, Köprunner M, Dorries J, Meyer D et al (2002) Guidance of primordial germ-cell migration by the chemokine SDF-1. Cell 111:647–659
Saito T, Goto-Kazeto R, Kawakami Y, Nomura K, Tanaka H, Adachi S et al (2011) The mechanism for primordial germ cell migration is conserved between japanese eel and zebrafish. PLoS One 6:e24460
Saito T, Goto-Kazeto R, Fujimoto T, Kawakami Y, Arai K, Yamaha E (2010) Inter-species transplantation and migration of primordial germ cells in cyprinid fish. Int J Dev Biol 54:1481–1486
Ciruna B, Weidinger G, Knaut H, Thisse B, Thisse C, Raz E et al (2002) Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc Natl Acad Sci U S A 99:14919–14924. https://doi.org/10.1073/pnas.222459999
Koprunner M, Thisse C, Thisse B, Raz E (2001) A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev 15:2877–2885
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Saito, T., Goto, R., Rivers, N., Yamaha, E. (2019). Production of Germ-Line Chimeras in Zebrafish. In: Pelegri, F. (eds) Vertebrate Embryogenesis. Methods in Molecular Biology, vol 1920. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9009-2_20
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DOI: https://doi.org/10.1007/978-1-4939-9009-2_20
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