雌二醇依赖性细胞稳定转染克隆株的研究

Stably Transfected Cell Lines for Detection of β Estradiol

关键词:  EGFP  表达载体  稳定转染细胞株

Key words:  EGFP  Expression vector  Stably transfected cell lines

摘要:

构建含报告基因的稳定转染的细胞株,为建立高通量筛选环境内分泌干扰物(EEDCs)的细胞表达系统打下基础。用本实验室已经构建以增强型绿色荧光蛋白(EGFP)为报告基因的表达载体和一个含有鸡溶菌酶基因超敏感位点4(hypersensitivity site 4)的载体pBS-HS₄,采用Polybrene的方法将它们进行稳定共转染T47D细胞得到稳定转染细胞株,用10_-9 M 17β-雌二醇诱导筛选。【结果】得到了87株稳定转染细胞株。标号为EGFP-T47D 74的克隆细胞株在诱导后72 h及96 h表现出非常强的诱导荧光,而且诱导倍数较大,为3.63倍。【结论】筛选出的稳定转染重组细胞株可供进一步的研究,以建立细胞表达系统来筛选EEDCs。

Abstract:

(Objective) The aim of this study was to obtain stably-transfected cell lines that could be used for developing high-throughput screening methods for EEDCs. (Method)A previously-constructed expression vector with enhanced GFP as reporter gene and pBS-HS₄ which included the hypersensitivity site 4 of chicken lysozyme gene were stably transfected into T47D cells using Polybrene methods. (Result) 87 stablely-tansfected cell clones were collected. When treated with 10_-9 M E2, Clone 74th showed very strong inducible fluorescence in the microscope after 72 hours and 96 hours induction and GFP expression achieved the highest fold induction (3.63) which determined by the Flx-800 fluorescence microplate reader. (Conclusion)The results indicated that the stable transfection cell lines could be used for further study to develop high-thoughput screenings method for EEDCs screening.