Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis)

A new cell line, CSEC, has been successfully established from embryos at gastrula stage of a cultured marine fish, half smooth tongue sole （Cynoglossus sernilaevis）. CSEC cells grow actively and stably more than 50 passages for over 200 d in DMEM medium supplemented with 15% FBS （fetal bovine serum）, 2.5 ng/cm^3 bFGF （basic fibroblast growth factor）, 1 ng/cm^3 LIF （leukemia inhibitory factor） and 50 mmol/dm^3 2-ME （2-mecaptoethanol）. The cells grew well in the temperature range of 24-30 ℃ and the optimal growth temperature was 24 ℃. FBS and bFGF concentrations are the two key components for CSEC cells proliferation. Chromosome analysis reveals that CSEC cells have a normal diploid karyotype with 2n=42t. The significant fluorescent signals were observed in CSEC after transfection with the GFP reporter gene, suggesting that the CSEC cell line can be used as a useful tool for transgenic and genetic manipulation studies. CSEC cells showed the cytopathic effect （CPE） after infection with lymphosystis disease virus （LCDV） in 2 d. Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy. It suggests that CSEC could be potentially used for the study of aquatic virus.

Half Smooth Tongue Sole(Cynoglossus semilaevis) Under Low Salinity Stress Can Change Hepatic igf2 Expression Through DNA Methylation

Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand the epigenetic regulation mechanism of igf2 under low salinity stress,we analyzed the DNA methylation at 5’UTR,exon1,intron1,and exon2,and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity(15)for 0,7,and 60 d.When exposed to low salinity,DNA methylation at 5’UTR and exon2 remained stable,while it was up-regulated firstly and then down-regulated at exon1 and intron1.Some single CpG sites of igf2 associated with low salinity,and most of these sites with significantly changed methylation levels(P<0.05)are located in intron1 area.The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity.Remarkably,the 38-CpG site of intron1 servers as a sexual marker.Additionally,our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression.The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress.Above all,to resist the low salinity stress,half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.