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Supplementary methods
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Xenogenic transplantation of primordial germ cells
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The transplantation of rainbow trout primordial germ cells (PGCs) was performed as
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previously reported (6). Briefly, donor PGCs labelled with green fluorescent protein (GFP)
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were prepared from excised genital ridges of pvasa-GFP transgenic rainbow trout fry, 35
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days post-fertilization (DPF), that were maintained in the Oizumi Research Station, Tokyo
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University of Fisheries (Yamanashi, Japan). After enzymatic dissociation, GFP-labelled
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PGCs were distinguished from gonadal somatic cells by their green fluorescence under a
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fluorescent dissecting microscope (SZX-12 with a SZX-RFL attachment and a GFP filter
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set, Olympus, Tokyo, Japan). Approximately 20 trout PGCs were injected into the
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peritoneal cavity of the 45 DPF newly hatched masu salmon fry, which were anaesthetized
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by 2-phenoxyethanol (Wako, Tokyo, Japan). The gonads of recipient embryos were
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observed under a fluorescent microscope (BX-50 with a BX-FLA attachment and a GFP
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filter set, Olympus) and photographed (using a DP-50, Olympus), in order to estimate the
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colonization rate of donor PGCs. All transplantation experiments used 20 embryos and
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were repeated three times; the data are represented as the mean ± standard error of the mean
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(SEM).
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Progeny test
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To determine the production of donor-derived spermatozoa, semen was collected from 1-
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year-old PGC-transplanted masu salmon. DNA was extracted from 1 l of semen and
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subjected to PCR with GFP-specific primers (5). The spermatozoa obtained from PCR-
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positive fish were used to fertilize rainbow trout eggs in a progeny test.
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DNA fingerprinting
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RAPD analysis was used to identify three different genotypes: rainbow trout, masu salmon
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and their hybrid. The genomic DNA of the masu salmon (lane 8) and a rainbow trout (lane
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9) were analysed as control. Genomic DNA samples were extracted from the trunk regions
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of individual fry. The PCR reaction mixture contained 0.2 g of template DNA, 0.25 units
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of Taq polymerase (Takara), 0.2 mM dNTPs and 1 M primers in a final volume of 10 l.
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The reaction was carried out for 35 cycles (94C for 1 min, 42C for 1 min and 72C for 2
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min), with a 3 min initial 94C denaturation step and a 5 min final elongation step. Next, 5
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l aliquots of the reaction mixtures were electrophoresed using a 1.2 % agarose gel that
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contained ethidium bromide. A total of 10 primers from a DNA oligomer set (Wako) were
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tested in the preliminary screening with two individuals from each species. One primer
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(TGGATTGGTC) was then selected for further analysis.
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