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目的 :建立一个可诱导表达目的基因的细胞系以研究新基因的功能。方法 :将含蜕皮素受体基因的表达载体pVgRXR稳定转染NIH3T3细胞 ,经zeocin抗性筛选 ,获得单克隆抗性细胞株 ,并进一步将可诱导表达质粒pIND_LacZ分别转染各单克隆细胞 ,经松甾酮 (ponasterone)A诱导和 β_半乳糖苷酶活性检测 ,鉴定出阳性单克隆。 结果与结论 :在建立稳定表达功能性蜕皮素受体的NIH3T3细胞系的基础上 ,我们进一步建立了一个可诱导表达红系分化相关基因 (EDAG)的细胞系 ,并对目的基因诱导表达特性及诱导目的基因表达后细胞表型的改变进行了初步观察 ,结果提示EDAG可能与血液细胞的增殖分化密切相关。
OBJECTIVE: To establish a cell line that can induce the expression of a gene of interest to study the function of the new gene. METHODS: NIH3T3 cells were stably transfected with the expression vector pVgRXR containing the ecdysone receptor gene and screened by zeocin to obtain monoclonal resistant cell lines. The inducible expression plasmid pIND_LacZ was transfected into each monoclonal cell separately. The results of ponasterone A induction and β-galactosidase activity detection showed that the monoclonal antibody was positive. RESULTS AND CONCLUSION: Based on the establishment of NIH3T3 cell line stably expressing functional ecdysone receptor, we further established a cell line that can induce the expression of erythroid differentiation-related gene (EDAG) Preliminary observation of the change of cell phenotype after induction of target gene expression suggests that EDAG may be closely related to the proliferation and differentiation of blood cells.