Establishment, Characterization Of A New Fish Astroglial Cell Line From The Brain Of Half-smooth Tongue Sole, Cynoglossus Semilaevis And Research Of A Sex Related Protein CSW3

The culture of fish cells can date back to1960s.Since theestablishment of the first fishcell line RTG-2, more than280fish cell lines have been established and researched inthe world. Plenty of fish organization have been used to establish fish cell lines,including kidney, liver, heart, fin, swim bladder, branchiostegal membrane, andembryo.The cell line devotes a system which is similar with in vivo environment,thus, it can be widely used in fish developmental biology, fish immunology, virology,toxicology, physiology,carcinogenesis, transgenic applications and other aspects.The half-smooth tongue sole (Cynoglossus semilaevis) has high economic value as amariculture fish specie in China. The half-smooth tongue sole has sexually dimorphicgrowth, with females growing faster than males. The tongue sole,which has42chromosomes and the ZW sex-determining system, has theretical and applical valuesin investigating the mechanisms of fish sexual growth dimorphism.Several cell lines from the spermary,ovary, head kidney, liver, heart and embryohave been established from the half-smooth tongue soleand widely used in virology,transgenic application. However, there is no establishment of astrogilial cell line inflatfish been reported before the CSAC. The CSAC will have broad applicationprospects in studying fish astrocyte characteristics and neural associated pathologicalmechanism.The CSAC was established, characterized and subcultured for more than57passages. MEM(Minimum Essential Medium Eagle) with20%FBS was optimum forthe growth of CSAC. The suitable growth temperature for CSAC was24oC.The cellidentity was tested by the presence of GFAP(glial fibrillary acidic protein)-theastroglial specifically expressed protein. RT-PCR and immunofluorescence revealed the expression of GFAP in the CSAC culture.Chromosome analysis revealed that thecells maintained a normal diploid chromosome number (2n=42) in the CSAC. Thefluorescent signals were observed in CSAC after the cells were transfected with greenfluorescent protein (GFP) reporter plasmids.The establishment of CSAC plays pivotalroles in researching the potential functions of fish astrogilal cells.The expression and application of a female specific gene CSW3were also studied inthis research, including the construction of CSW3recombinant expression vector andtransformation to E.coli. The expression protein was isolated and purified. The femalespecific CSW3protein can cause female related gene foxl2up-regulation and malerelated gene sox9a and amh decreasion.