Reproductive Technology

Green Fluorescent Protein Labeling of Primordial Germ Cells Using a Nontransgenic Method and Its Application for Germ Cell Transplantation in Salmonidae

Abstract
Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa-gene regulatory region. However, for PGC-transplantation to be practically useful in aquaculture, visualization of PGCs using a non-transgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa-gene 3'-untranslated regions (UTR); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. GFP chimeric RNAs including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish were microinjected into the cytoplasm of fertilized eggs of several salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somitogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC-transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor-PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This non-transgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate broodstock technology.

Key words: Embryo • Developmental biology • Early development • Gametogenesis • Gene regulation