Xenogenesis in Teleost Fish Through Generation of Germ-Line Chimeras by Single Primordial Germ Cell Transplantation1

Abstract

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

“…To exclude the influence of somatic cells while simultaneously ensuring that we transplanted PGCs, we isolated PGCs from embryos that had been injected with GFP-nos1 3'UTR mRNA to label the germ cells. We previously showed that a single PGC isolated from a 10-15-somite stage could migrate toward the gonadal region of a host embryo after transplantation (Saito et al, 2008). However, in the earlier study, we did not determine the optimal stage of donor embryo development for isolating PGCs for successful SPT.…”

“…In many host embryos, the transplanted PGCs successfully migrated toward gonadal anlage. These transplantation experiments showed that viable sperm could be produced in germ-line chimeras between different species, that is, xenogenesis was feasible across species, genus, and family barriers (Saito et al, 2008). In similar fashion, it has been reported that intra-specific germ-line chimeras can be produced by transplanting blastomeres at the blastula stage (Lin et al, 1992;Ciruna et al, 2002;Yamaha et al, 2001;Yamaha et al, 2003;Takeuchi et al, 2001).…”

“…Transplantation of germ cells from a donor into a genetically different host produces a germ-line chimera. This concept underlies recent attempts to obtain viable gametes from germ-line chimeras (Brinster and Zimmermann, 1994;Brinster and Avarbock, 1994;Clouthier et al, 1996;Yamaha et al, 2001;Yamaha et al, 2003;Takeuchi et al, 2001;Takeuchi et al, 2003;Takeuchi et al, 2004;Saito et al, 2008).…”

“…Subsequently, gonadal somatic cells of the host surround the transplanted germ cells; this reorganization may disable any later immunological rejection of the germ cells. It has been shown in species such as zebrafish that germ cells can be transplanted at early embryonic stages (Saito et al, 2008). In this study, donor PGCs were visualized with GFP by injecting GFP-nos1 3'UTR mRNA into one-to four-cell stage embryos.…”

Learning to Fish with Genetics: A Primer on the Vertebrate ModelDanio rerio

“…Females can also be mated to WT males to produce clutches of embryos without maternal gene products. Germ-line replacement has now been used on a wide variety of fish species and even in cross-species transplants, opening up additional opportunities for research on gamete development (Saito et al 2008;Shimada and Takeda 2008;Goto et al 2012).…”