蜕皮素诱导基因表达细胞系的建立及其应用

Methods for establishing stable ecdysone-inducible cell line

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 stable cell line that can be induced to express a target gene.Methods:The expression vector pVgRXR containing the gene of ecdysone receptor was transfected into NIH3T3 cells, and monoclonal cell lines were acquired by zeocin resistant selection, which stably express functional heterodimeric ecdysone receptors. Then, these monoclonal cell lines were transfected with pIND/LacZ, an inducible expression plasmid, and the positive monoclonal cell lines were identified by induction of ponasterone A and detection of β-galactosidase activity. Furthermore, the inducible expression characteristic of target gene and the alteration of cell phenotype after induced expression of target gene were investigated.Results and Conclusion:On the basis of NIH3T3 cell line stably expressing functional ecdysone receptors, a new cell line inducibly expressing erythrocyte development associated gene (EDAG) was established. And the results indicated that EDAG might be closely associated with the proliferation and differentiation of blood cells.