Use of cultured primordial germ cells for production of transgenic fish

摘要

Due to its favorable characteristics, the zebrafish is a popular model of vertebrate development. However, one deficiency of the zebrafish model system is the lack of methods for cell-mediated gene transfer and targeted mutagenesis. In mice, cell-mediated gene transfer is accomplished through the use of embryonic stem (ES) cell cultures and provides the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. ES cells possessing a targeted mutation are selected in culture and transferred to a host embryo. Transgenic mice possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of the chimeric embryo. In zebrafish, embryo cell cultures have beenderived that exhibit in vitro characteristics of ES cells but 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, maintained in the presence of cells from a rainbow trout spleen cell line, RTS34st, are able to produce germ line chimeras when introduced into a host embryo. Zebrafish embryo cells co-cultured with RTS34st cells or their conditioned medium continue to possess mRNAencoding the primordial germ cell marker, vasa, for more than 30 days. In the absence of RTS34st cells or conditioned medium the vasa mRNA disappeared by five days in culture. The spleen cells also inhibited the embryo cell cultures from differentiatinginto melanocytes and neuronal cell types. The influence of the RTS34st cells on the zebrafish embryo cell cultures indicate that the splenic stromal cell line will be a valuable tool in the application of cell-mediated gene transfer and targeted gene inactivation technology to zebrafish.