Abstract
To develop a cell-mediated gene transfer technique in fish, a cell line that can differentiate into the germ cell lineage is needed. In this study, we developed techniques to identify, visualize, and isolate live primordial gems cells (PGCs) for in vitro culture and later for gene transfer work. In many animals, the vasa gene products have been found in the germ cell lineage, and we have previously identified and characterized its homologue (RtVLG) in rainbow trout. Whole mount in situ hybridization of trout embryos at various developmental stages revealed that transcripts were localized to PGCs, suggesting that RtVLG transcription can be used as a marker for PGCs. To identify PGCs in vivo, we constructed a plasmid containing the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (pvasa-GFP), microinjected it into fertilized eggs, and raised mature fish. The F1 generation of these transgenic trout showed green fluorescence only in PGCs in all developmental stages. This expression pattern was consistent with that of the endogenous RtVLG gene. To obtain PGCs for in vitro culture, the genital ridges were excised from transgenic embryos, dissociated by trypsin, and a flow cytometer was used to sort them into GFP-positive and GFP-negative fractions. RT-PCR analysis revealed that RtVLG was expressed only in GFP-positive cells. These results confirm that the isolated GFP-positive cells are PGCs. We are presently attempting to establish cell lines for further work.
