Cell culture and transfection

The medaka haploid ES cell line HX1 was maintained at 28°C in the medium of ESM4 as previously described (Hong and Schartl, 2006; Yi et al., 2010). The grouper spleen cell line GS was maintained at 25°C in L15-medium (Leibovitz) containing 10% fetal bovine serum (Huang et al., 2009). Cell transfection was performed by using DNAfectin reagent (Applied Biological Materials, Richmond, BC, Canada) essentially as described (Hong et al., 2004a). Briefly, 2 µg of plasmid DNA (p88GFP or pGFP) and 8 µL of DNAfectin reagent were mixed in 200 µL of pure DMEM. After incubation at room temperature for 20 min, the transfection mixture was added dropwise to cells in a 6-well plate containing 2 mL of DMEM. After incubation for 6 h at 28°C, the cells were grown in ESM4 for 48 h and subcultured in 10-cm dishes for clonal growth in the presence of 0.5 mg/mL of G418 (Hong et al., 1996). The medium was changed every 5–7 days. Single colonies comprising GFP-positive cells were picked with 200-µL tips into 96-well plates and serially expanded into 88GFP-HX1 cells (p88GFP transfectants) and GFP-HX1 cells (pGFP transfectants) as described (Hong et al., 1996).