Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation

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.

原始生殖细胞（PGCs）是发育中胚胎里唯一有潜力将遗传信息传递给下一代的细胞。因此，PGCs在基因库构建和低温保存方面具有潜在价值，尤其是通过利用生殖系嵌合体生产供体配子来实现。目前，尚不清楚生殖系分化和性腺结构形成需要多少PGCs。在本研究中，我们通过向每个受体胚胎移植单个PGC，实现了两种亲缘关系较近的硬骨鱼物种——珍珠丹鱼（Danio albolineatus）和斑马鱼（Danio rerio）之间的完全生殖系替代。我们分离出单个PGC并将其移植到处于囊胚期的斑马鱼胚胎中。通过反义“死胡同”吗啉代寡核苷酸来阻止受体生殖系细胞的发育。在许多受体胚胎中，移植的供体PGC在不进行细胞分裂的情况下成功迁移至性腺原基。在性腺原基处，PGC分化形成一个正常大小的性腺，而非通常的一对性腺。通过这些嵌合体的自然产卵获得了后代。形态学和DNA分析表明，这些后代源自供体。我们进一步拓展研究，证实移植到斑马鱼受体胚胎中的金鱼（Carassius auratus）和泥鳅（Misgurnus anguillicaudatus）的单个PGC同样能够分化为精子。我们的研究结果表明，异种生殖跨越物种、属和科的界限不仅具有现实可能性，而且通过移植供体物种的单个PGC即可实现。