Establishment of a pluripotent embryonic cell line from sea perch ( Lateolabrax japonicus) embryos

Abstract

Pluripotent embryonic stem (ES) cells provide an efficient approach for genome manipulation with many applications in marine biotechnology and development studies. To develop this technology, we have worked to derive fish ES cells for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. A pluripotent cell line, LJES1, was established from blastula-stage embryos of a cultured marine fish, Lateolabrax japonicus. The LJES1 cells were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with fetal bovine serum (FBS), marine fish serum, sea perch embryo extract, selenium, basic fibroblast growth factor (bFGF) and leukemia inhibitory factor. The ES-like cells were small and round or polygonal, and grew actively and stably in culture. The cells exhibited a positive alkaline phosphatase activity upon histochemical staining. When the cells were treated with all- trans retinoic acid, differentiation into various types of cells, including neuron-like cells, muscle cells and some unidentified cells were observed, suggesting that the LJES1 cells remained pluripotent in culture. Chromosome analysis revealed that LJES1 cells have a normal diploid karyotype with 2 n=48. Up to now, LJES1 cells have been continuously cultured for more than 150 days with more than 40 passages. High survival rate has been obtained after cryopreservation of cell cultures. GFP reporter gene were transferred into LJES1 cells and successfully expressed.