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Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis) 总被引：2，自引：1，他引：2
A new cell line,CSEC,has been successfully established from embryos at gastrula stage of a cultured marine fish,half smooth tongue sole(Cynoglossus semilaevis).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. 相似文献
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Chen Gang 《中国海洋大学学报(自然科学版)》2000,30(Z1):19-24
以CTAB法提取含有番茄黄化曲叶病毒TYLCV-CHI病原的烟草曲 叶病烟草DNA为模板,扩增该病毒外壳蛋白启动子序列,克隆到pGEM7Z载体上,在启动子下 游连接上能表达毒素蛋白的RNase基因和终止子NOS,最终将构建的基因转移到带有绿色荧光 蛋白GFP的植物表达载体上,构建了通过病毒侵染后激发病毒外壳蛋白启动子从而表达毒素 基因,产生毒素杀死病毒防止感染继续扩散的自我保护机制的基因工程植物表达载体。 相似文献
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A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco’s modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies. 相似文献
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为探讨迟缓爱德华菌(Edwarsiellatarda)入侵途径,建立感染模型,作者通过电转化法构建GFP标记的迟缓爱德华菌EtMc1512(质粒PMDpp-EGFP),实验设立浸泡组、腹腔注射组和肌肉注射组,感染后采集各组实验诸氏鲻虾虎鱼(Mugilogobius chulae)血液、鳃、肝脏、肠、肌肉,培养法统计分析各组织中的荧光细菌数;浸泡组取样时间为0、2、4、6、8、12、24 h,腹腔注射组和肌肉注射组取样时间为6、12、24、48、72、96h。结果显示,构建的EtMc1512-GFP具有较强荧光,GFP标记前后菌株毒力基因(citC、mukF、esrB、katB、fimA、gadB)检测结果均为阳性。浸泡感染后实验鱼各组织内的荧光菌随时间表现为先升后降的趋势,最高菌量出现在肠道(2.51×106CFU/g),其次为鳃(4.19×104CFU/g)、血液(1.65×104CFU/g),肠道荧光菌显著高于其他组织(P0.05);腹腔注射感染后肝脏(4.55×106CFU/g)和血液(4.65×106CFU/g)菌量最高;肌肉注射感染后肌肉在48h首先检出荧光菌,血液(2.93×104 CFU/g)菌量最高。结果表明,肠道、肝脏和肌肉分别是迟缓爱德华菌浸泡感染、腹腔注射感染和肌肉注射感染诸氏鲻虾虎鱼的主要组织器官,在自然条件下迟缓爱德华菌经口感染诸氏鲻虾虎鱼风险较高。 相似文献
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To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii,a high efficient expression vector was constructed.Green fluorescent protein(GFP) was expressed in C.reinhardtii under the control of promoters:RBCS2 and HSP70A-RBCS2.Efficiency of transformation and expression were compared between two transgenic algae:RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ.Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2,and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ.In addition,a threefold increase of GFP in Tran-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii. 相似文献
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Characteristics of ChgH–GFP transgenic medaka lines, an in vivo estrogenic compound detection system
We previously reported the characteristics of a ChgH–GFP transgenic medaka line that indicates estrogenic compound pollution in environmental water by the green fluorescence of their liver. Recently, we established four more lines. In this study, the characteristics of the five transgenic medaka lines were investigated. The intensity of reporter gene expression varied among transgenic lines and generally correlated well with the amount of integrated transgene in each line. Line-specific ectopic expression was also observed. However, the sensitivity to 17-β estradiol did not differ among transgenic lines. Three transgenic lines are considered to be suitable as bio-indicators of estrogenic activity, due to the ease of observing green fluorescence in their livers. The transgenic lines can also detect the estrogenic activity of testosterone and 17-β trenbolone at the nominal concentration of 30 and 100 μg/l, respectively. 相似文献
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To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii. 相似文献
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The ovary is an excellent system for studying stem cell renewal and differentiation, which is under the control of ovarian somatic cells. In order to understand oogenesis in Fugu rubripes (Temminck et. Schlegel) as a marine fish model of aquaculture importance, we established cell lines called TSOC1 and TSOC2 from a juvenile ovary of this organism. TSOC1 is composed of spindle epithelial-like cells, while the other is cobblestone-like cells. Therefore, TSOC1 and TSOC2 appear to consist of ovarian somatic cells. Growth requirement condition was investigated including temperature, concentration of FBS and pH. Significant fluorescent signals were observed after TSOC1 and TSOC2 cells were transfected with pEGFP-N3 vector, indicating its potential utility for genetic manipulation such as gene function studies. It is shown that these cell lines are effective for infection by the turbot reddish body iridovirus and flounder lymphosystis disease virus as evidenced by the appearance of cytopathic effect and virus propagation in the virus-infected cells, and most convincingly, the observation of viral particles by electron microscopy, demonstrating that TSOC1 and TSOC2 are suitable to study interactions between virus and host cells. It is believed that TSOC1 and TSOC2 will be useful tools to study sex-related events and interactions between primordial germ cells and oogonia cells during oogenesis. Therefore, establishment of ovary cell lines from Fugu rubripes seems to be significant for those research areas. 相似文献
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