Establishment of a novel clonal GFP-expressing transgenic ginbuna crucian carp

The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is suitable for cell transplantation studies to reveal the roles of stem cells and immune cells. To ensure long-term traceability of donor cells within recipient fish, we have established a transgenic ginbuna line that expresses green fluorescent protein (GFP). The Xenopus laevis ef1a promoter was introduced for regulating GFP expression. Tol2 transposon-based transgenesis to ginbuna embryos resulted in producing a putative founder fish (F0) in a mosaic fluorescent fashion; the frequency of germline transmission was 14.9%. All embryos of GFP-positive offspring (F1)-derived F2 generation expressed GFP widely across the body. The result of Southern blot analysis showed that the transgene was present on a single DNA fragment of equivalent size among F1 and F2 individuals tested, indicating that the transgene was stably transmitted without translocation. Analysis of the fluorescence intensity of organs obtained from F1 and F2 juveniles using fluorescence microscope showed that eyes, brain, skeletal muscle, heart and gonad exhibited a strong GFP fluorescence while gill, spleen and intestine gave a weak signal; no fluorescence was observed in erythrocytes. Flow cytometric analyses of peripheral leukocytes from F1 and F2 adult fish revealed all cell populations expressed GFP. Scale grafts from the transgenic fish to the wild-type fish exhibited persistent engraftment. Together, our transgenic line can be a powerful tool for studying cellular dynamics by cell transplantation and provide a solid basis for further immunological research advances in teleost.