A cell line, SHK, was derived from the kidney of spotted halibut Verasper variegates. The cell line was subcultured more than 40 passages in minimum essential medium （MEM） supplemented with fetal bovine serum （FBS...A cell line, SHK, was derived from the kidney of spotted halibut Verasper variegates. The cell line was subcultured more than 40 passages in minimum essential medium （MEM） supplemented with fetal bovine serum （FBS） and 10 ng ml4 basic fibroblast growth factor （bFGF）. Cell morphology from primary culture and subculture was observed continuously by microscopy. The SHK cell line consisted predominantly of fibroblast-like cells. The cell line was able to grow between 20℃ and 30℃ with the optimum growth at 24℃ and with a reduced growth between 12℃ and 20℃. The growth rate of the cells increased as the proportion of FBS increased from 10% to 20% at 28℃ with optimum growth at the concentration of 20%. The doubling time of the cells was determined to be 44.8 h. Chromosome analysis revealed that 52% of the SHK cells maintained a normal diploid chromosome number （2n=46）. The cells were successfully transfected with green fluorescent protein （GFP） reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. The cells were infected by lymphosystis disease virus （LCDV） and found to be susceptible to the virus in cytopathic effect （CPE） observation. The infection was confirmed by PCR and electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.展开更多
A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew...A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco's modified Eagle medium/F12 medium(DMEM/F12, 1:1; p H 7.2) supplemented with 20% fetal bovine serum(FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor(b FGF) and insulin-like growth factor-I(IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number(46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein(GFP) positively expressed in these cells after being transformed with pc DNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.展开更多
Turbot(Scophthalmus maximus L.) reddish body iridovirus(TRBIV) was propagated in turbot fin cells(TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study.TF cells were cul...Turbot(Scophthalmus maximus L.) reddish body iridovirus(TRBIV) was propagated in turbot fin cells(TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study.TF cells were cultured in 10% bovine calf serum(BCS)-containing MEM medium(pH7.0) at 22℃,in which TRBIV propagated to a titer as high as 105.6 TCID50 mL-1.The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide.The inactivated vaccine caused neither cytopathogenic effect(CPE) on TF cells nor pathogenic effect on turbots.After being administered with the vaccine twice via muscle injection,the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner.The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms,respectively.The inactivated vaccine was very immunogenic,efficiently preventing tur-bot from death.It holds the potential of being applied in aquaculture.展开更多
Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitabl...Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 individual fish.The number of alleles ranged from 2 to 11, with an average of 4.614 alleles per locus.The values of observed and expected heterozygosities ranged from 0.1000 to 1.0000 and from 0.0966 to 0.8847 respectively. Polymorphism information content (PIC) ranged from 0.0905 to 0.862.These markers would be useful for population structure assessment,genetic linkage map construction and parentage analysis for this species.展开更多
To develop a rabbit corneal endothelial(RCE)cell line,in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study.To initiate the primary cult...To develop a rabbit corneal endothelial(RCE)cell line,in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study.To initiate the primary culture of RCE cells,corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down.After being cultured at a time-gradient interval from 48 to 6 h,the corneal slices were detached and reattached into new wells,respectively.Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20%FBS-DMEM/F12 medium con-taining chondroitin sulfate,ocular extract,epidermal growth factor(EGF),basic fibroblast growth factor(bFGF),carboxymethyl-chitosan,N-acetylglucosamine hydrochloride,glucosamine hydrochloride,culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul-fate at 37℃,5%CO_(2).The cultured RCE cells,in quadrangle and polygonal shapes,proliferated to con-fluence 3 weeks later.During the subsequent subculture,the shape of RCE cells changed gradually from polygonal to more fibroblastic.A novel RCE cell line,growing at a steady rate,with a population doubling time of 53.8 h,has been established and subcultured to passage 67.Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44.The results of immuno-cytochemical staining with neuron specific enolase(NSE)confirmed that the RCE cells were in neuroectodermal origin.Combined with the results of vascular endothelial growth factor(VEGF)treatment and endothelial cell morphology recovery,it can be concluded that the cell line established here is an RCE cell line.This RCE cell line may serve as a useful tool in theoretical re-searches of mammalian corneal endothelial cells,and may also have potential application in artificial corneal endothelium development.展开更多
基金supported by grants from State 863High-Technology Rand Project of China(2006AA09Z406,2006AA10A401)Taishan Scholar Project of Shan-dong Province
文摘A cell line, SHK, was derived from the kidney of spotted halibut Verasper variegates. The cell line was subcultured more than 40 passages in minimum essential medium （MEM） supplemented with fetal bovine serum （FBS） and 10 ng ml4 basic fibroblast growth factor （bFGF）. Cell morphology from primary culture and subculture was observed continuously by microscopy. The SHK cell line consisted predominantly of fibroblast-like cells. The cell line was able to grow between 20℃ and 30℃ with the optimum growth at 24℃ and with a reduced growth between 12℃ and 20℃. The growth rate of the cells increased as the proportion of FBS increased from 10% to 20% at 28℃ with optimum growth at the concentration of 20%. The doubling time of the cells was determined to be 44.8 h. Chromosome analysis revealed that 52% of the SHK cells maintained a normal diploid chromosome number （2n=46）. The cells were successfully transfected with green fluorescent protein （GFP） reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. The cells were infected by lymphosystis disease virus （LCDV） and found to be susceptible to the virus in cytopathic effect （CPE） observation. The infection was confirmed by PCR and electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.
基金supported by grants from the National 863 High Technology Research Foundation of China(2006AA10A401)the National Natural Science Foundation of China(31001100)
文摘A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco's modified Eagle medium/F12 medium(DMEM/F12, 1:1; p H 7.2) supplemented with 20% fetal bovine serum(FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor(b FGF) and insulin-like growth factor-I(IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number(46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein(GFP) positively expressed in these cells after being transformed with pc DNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.
基金supported by the National High Technology Research and Development Program of China(863 Program)(2006AA10A401)
文摘Turbot(Scophthalmus maximus L.) reddish body iridovirus(TRBIV) was propagated in turbot fin cells(TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study.TF cells were cultured in 10% bovine calf serum(BCS)-containing MEM medium(pH7.0) at 22℃,in which TRBIV propagated to a titer as high as 105.6 TCID50 mL-1.The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide.The inactivated vaccine caused neither cytopathogenic effect(CPE) on TF cells nor pathogenic effect on turbots.After being administered with the vaccine twice via muscle injection,the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner.The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms,respectively.The inactivated vaccine was very immunogenic,efficiently preventing tur-bot from death.It holds the potential of being applied in aquaculture.
基金supported by the 863 Project of China(2006AA10A403)the Natural Science Foundation of China(No.30972244)Taishan Scholar Project of Shandong Province,China
文摘Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 individual fish.The number of alleles ranged from 2 to 11, with an average of 4.614 alleles per locus.The values of observed and expected heterozygosities ranged from 0.1000 to 1.0000 and from 0.0966 to 0.8847 respectively. Polymorphism information content (PIC) ranged from 0.0905 to 0.862.These markers would be useful for population structure assessment,genetic linkage map construction and parentage analysis for this species.
基金863 High-Tech Research and Development Program of China(Grant No.2001AA625050)
文摘To develop a rabbit corneal endothelial(RCE)cell line,in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study.To initiate the primary culture of RCE cells,corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down.After being cultured at a time-gradient interval from 48 to 6 h,the corneal slices were detached and reattached into new wells,respectively.Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20%FBS-DMEM/F12 medium con-taining chondroitin sulfate,ocular extract,epidermal growth factor(EGF),basic fibroblast growth factor(bFGF),carboxymethyl-chitosan,N-acetylglucosamine hydrochloride,glucosamine hydrochloride,culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul-fate at 37℃,5%CO_(2).The cultured RCE cells,in quadrangle and polygonal shapes,proliferated to con-fluence 3 weeks later.During the subsequent subculture,the shape of RCE cells changed gradually from polygonal to more fibroblastic.A novel RCE cell line,growing at a steady rate,with a population doubling time of 53.8 h,has been established and subcultured to passage 67.Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44.The results of immuno-cytochemical staining with neuron specific enolase(NSE)confirmed that the RCE cells were in neuroectodermal origin.Combined with the results of vascular endothelial growth factor(VEGF)treatment and endothelial cell morphology recovery,it can be concluded that the cell line established here is an RCE cell line.This RCE cell line may serve as a useful tool in theoretical re-searches of mammalian corneal endothelial cells,and may also have potential application in artificial corneal endothelium development.
