Establishment and characterization of a gonad cell line from half-smooth tongue sole Cynoglossus semilaevis pseudomale.
A new cell line was established from half-smooth tongue sole Cynoglossus semilaevis pseudomale gonad (CSPMG). Primary culture was initiated from gonad tissues pieces, and the CSPMG cells were cultured at 24 °C in Dulbecco's modified Eagle medium/F12 medium (1:1) (pH7.0), supplemented with 20 % fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, 2-mercaptoethanol, penicillin and streptomycin. The cultured CSPMG cells, in fibroblast shape, proliferated to 100 % confluency 10 days later and had been subcultured to passage 109. Chromosome analyses indicated that the CSPMG cells exhibited chromosomal aneuploidy with a modal chromosome number of 42, which displayed the normal diploid karyotype of half-smooth tongue sole (2n = 42t, NF = 42). Reverse transcription polymerase chain reaction revealed CSPMG cells could express gonad somatic cell functional genes Sox9a, Wt1a and weakly germ cell marker gene Vasa, but not male specific gene Dmrt1. Transfection experiment demonstrated that CSPMG cells transfected with pEGFP-N3 plasmid and small RNA could express green and red fluorescence signals with high transfection efficiency. In conclusion, a continuous CSPMG cell line has been established successfully. The cell line might serve as a valuable tool for studies on the mechanism of sex determination, sex reversal and gonad development in flatfish.
Key Lab of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China.
Ai
A
Sun
Song-Lin
SL
Chen
Feng-Tao
FT
Gao
Hai-Long
HL
Li
Xiao-Feng
XF
Liu
Na
N
Wang
Zhen-Xia
ZX
Sha
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods / Q000662:veterinary*; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D003470:Culture Media / Q000737:chemistry*; D020732:Cytogenetic Analysis / Q000662:veterinary; D004815:Epidermal Growth Factor / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry; D005412:Flatfishes; D006066:Gonads / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry; D010406:Penicillins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D055758:SOX9 Transcription Factor / Q000378:metabolism; D013002:Somatomedins / Q000737:chemistry; D013307:Streptomycin / Q000737:chemistry; D025721:WT1 Proteins / Q000378:metabolism; D000097573:Red Fluorescent Protein
D003470:Culture Media; D004815:Epidermal Growth Factor; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008623:Mercaptoethanol; D010406:Penicillins; D055758:SOX9 Transcription Factor; D013002:Somatomedins; D013307:Streptomycin; D025721:WT1 Proteins; D000097573:Red Fluorescent Protein