Production of zebrafish germ-line chimeras from embryo cell cultures

Although the zebrafish possesses many characteristics that make it a valuab le model for genetic studies of vertebrate development, one deficiency of t his model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are r outinely used for gene transfer and provide the advantage of in vitro selec tion for rare events such as homologous recombination and targeted mutation . Transgenic animals possessing a mutated copy of the targeted gene are gen erated when the selected cells contribute to the germ line of a chimeric em bryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures m aintained in the presence of cells from a rainbow trout spleen cell line (R TS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vase, was p resent for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cel l differentiation in the embryo cell cultures. These results suggest that t he RTS34st splenic-stromal cell line will be a valuable tool in the develop ment of a cell-based gene transfer approach to targeted gene inactivation i n zebrafish.