Homologous recombination and directed differentiation in medaka ES cells: Development of vector systems

Abstract

The combination of medaka embryonic stem cell and bicistronic plasmid system make it a good platform to study various molecular and cellular events. We first developed a single plasmid system for fast assay of relative cellular activities for homologous recombination versus random integration. In this system, a cell that has undergone a homologous recombination has red cytoplasm, while cell with a nonhomologous recombination possesses a green nucleus. The roles of important DNA-repairing genes Rad51 and Dmc1 in this cellular event were studied by ectopic expression. Then we took directed melanocyte differentiation as a model to enrich transgenic differentiating cells. For this, a bicistronic plasmid was constructed that enables the transgenic medaka embryonic stem cell to differentiate into melanocyte and confer drug resistance and green fluorescence as well. Highly enriched differentiating melanocytes were obtained using this system by drug selection.