Establishment And Premilinary Applications Of Cell Lines From Epinephelus Moara

The research of fish cell culture started in 1960. The establishment of fish cell line and the correlational research made progress rapidly since 1962 when Wolf and Quimby established the first fish cell line RTG-2. Fish tissues used to establish fish cell lines were acquired from embryo, brain, bone, muscle, fin, eye, gill, tumor, viscera and so on. Fish cell lines had used in virology, toxicology, physiology, transgenic applications, carcinogenesis, cytoskeleton, repair of DNA damage and other aspects.Epinephelus moara, a commercially valuable and fast growing marine fish species with good quality in nutrients and taste, is an important aquaculture species in China. Recently, outbreak of iridovirus and other viruses has caused great economic losses in aquaculture. The Epinephehis moara is a protogynous hermaphroditic fish that can be used as an ideal material for research of sex reversal.In this paper, three new cell lines, EMH, EMB and EMK, were established from heart, brain and kidney of Epinephelus moara. The cell lines were maintained in Leibovitz’s L15 medium supplemented with HEPES, antibiotics, fetal bovine serum (FBS),2-Mercaptoethanol (2-ME), and basic fibroblast growth factor (bFGF).The cultured EMH cells, fibroblastic and epithelial in morphology, proliferated to 100% confluency 4-5 days later and had been subcultured more than 20 passages. The suitable temperature for the cell growth was 18℃～30℃ with the optimum growth at 30℃ and an unfavorable growth at 36℃. The suitable FBS concentration for the cell growth was 10%～20% with the optimum concentration at 15%. Chromosome analysis revealed that 62% of the cells maintained a normal diploid chromosome number (2n=48) in the EMH cells at Passage 20.The cultured EMB cells, fibroblastic in morphology, proliferated to 100% confluency 3 days later and had been subcultured more than 60 passages. The suitable temperature for the cell growth was 18℃～30℃ with the optimum growth lying between 24℃～30℃. The suitable FBS concentration for the cell growth was 10%-20% with the optimum concentration at 15%. Chromosome analysis revealed that 56% of the cells maintained a normal diploid chromosome number (2n=48) in the EMB cells at Passage 45.The cultured EMK cells, fibroblastic in morphology, proliferated to 100% confluency 3-4 days later and had been subcultured more than 50 passages. The suitable temperature for the cell growth was 18℃～30℃ with the optimum growth at 24℃. The suitable FBS concentration for the cell growth was 10%～20% with the optimum concentration at 15%. Chromosome analysis revealed that 58% of the cells maintained a normal diploid chromosome number (2n=48) in the EMK cells at Passage 42.The fluorescent signals were observed in EMH, EMB and EMK cells after the cells were transfected with enhanced green fluorescent protein (EGFP) reporter plasmids. The EMH, EMB and EMK established here can be used as useful tools for transgenic and may serve as potential applications in fish virus isolation, propagation and vaccine development.