A transgenic medaka line with visible markers for genotypic and phenotypic sex

Accurate genotyping of sex is required for correct interpretation in any in vivo assays with endocrine disrupting chemicals (EDCs). Visible markers for genotypic sex, if reliable, simplify assays because time-consuming PCR-based genotyping can be skipped. Here, we describe a line of Japanese medaka with a brain-expressed green fluorescent protein (GFP) transgene inserted near the sex-determining locus. When used with a white pigment cell marker, genotypic sex can be determined reliably as early as 3 days after fertilization (well before gonadal sex differentiation). No recombinants were found in more than 2000 progenies. We also introduced a strong ovarian GFP marker into the line with these genetic sex markers, so that phenotypic sex can also be determined reliably at 8 days after hatching. Well-known sex reversal protocols using exogenous steroid treatments of embryos were monitored by this transgenic line, demonstrating the line to be a useful tool for in vivo studies utilizing gonadal sex differentiation of the medaka, especially for screenings of potential estrogenic and androgenic EDCs.