Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Onc orhynchus mykiss, by microinjection of the isolated blastomeres. FITC-label ed donor cells and non-labeled recipient embryos at various developmental s tages between the early blastula and early gastrula stages were used for ce ll transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient e mbryos, the combination of midblastula (2.5 dpf) donor cells and early blas tula (1.5 dpf) recipient embryos gave the highest chimera formation rate an d the best distribution pattern of donor cells. Using this combination, chi meric rainbow trout were produced with donor blastomeres from dominant oran ge-colored mutant embryos and wild-type recipient embryos. Of the 238 chime ric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized wit h gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. Th e contribution rates of donor cells in the germ-line ranged from 0.3 to 14% . This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body colo r mutants are used for either donor cells or the host embryos, it will be p ossible to easily concentrate FI transgenic embryos derived from transplant ed donor cells by body color screening, (C) 2001 Wiley-Liss, Inc.