Identification of promoter elements responsible for gonad-specific expression of zebrafish Deadend and its application to ovarian germ cell derivation.

We discovered that a 150-bp region of zebrafish deadend (dnd) spanning the translation start codon, exon 1 and part of intron 1 is required to direct heterologous neomycin-resistance gene (neo) expression specifically in the gonad, similar to endogenous dnd. Using an 8.3-kb dnd promoter that contains this 150-bp region, we generated Tg(dnd:neo-dnd) transgenic zebrafish in which the expression of Neo was detected specifically in ovarian germ cells. The transgenic fish were used to initiate primary ovarian germ cell cultures with antibiotic G418 to select ovarian germ cells and eliminate ovarian somatic cells. RT-PCR results demonstrated that the drug-selected ovarian germ cells continued to express germ-cell markers nanos3, vasa and dnd. Growth assays demonstrated that recombinant zebrafish Lif had a significant mitogenic effect on the ovarian germ cells. When long-term ovarian germ cell cultures were transplanted into two-week-old infertile larvae, they successfully colonized and directed the formation of a truncated gonad in the recipient adult fish. Histological examination of the recipient adult fish revealed that 9 out of 34 individuals (26%) possessed donor-derived cells in their gonads. The identification of zebrafish dnd promoter and the use of this promoter to generate Tg(dnd:neo-dnd) led to the success of germ cell isolation through drug selection to generate homogenous germ cells that can be used to study zebrafish germ cell biology and may lead to a cell-mediated gene transfer strategy.