Derivation and characterization of zebrafish embryonic germ cell cultures and the effects of kit ligand a

A stem cell-mediated gene targeting approach is currently not available for zebrafish. To address this problem our lab has developed methods for the culture of pluripotent stem cells derived from early-stage zebrafish embryos. Embryonic stem (ES) cell cultures were derived from blastula-stage zebrafish embryos and primary cultures of pluripotent embryonic germ (EG) cells were initiated from primordial germ cells (PGCs). To be useful for gene targeting studies, optimization of the culture conditions is required to maintain the stem cells in a pluripotent condition for multiple passages. Also methods must be established to introduce targeting plasmids into the cells by homologous recombination and selection strategies are needed to isolate colonies of cells that have undergone the targeting event. In this study, I have developed methods to initiate multiple-passage EG cell cultures derived from zebrafish PGCs. A transgenic approach involving drug selection was used to isolate homogeneous populations of PGCs for cell culture. Optimization of the PGC culture conditions using recombinant zebrafish growth factors demonstrated that Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b) promoted the in vitro growth of the PGCs. Although PGCs do not express kit receptor in vivo, in culture the cells expressed the receptor and responded to recombinant zebrafish Kitlga added to the medium. After several days in the presence of these factors, the PGC cultures began to exhibit in vitro characteristics of pluripotent EG cells. Kitlga was especially important for promoting the expression of pluripotency markers including nanos, alkaline phosphatase and SSEA-1. The EG cells also began to express endogenous kitlga and inhibition of its expression or function with antisensense morpholinos or MAP kinase inhibitors demonstrated that the factor was important for maintaining pluripotency. In a separate project, gene targeting methods were established for zebrafish ES cell cultures. The results of these studies demonstrated that the ES cells are able to incorporate plasmid in a targeted fashion by homologous recombination. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene