Inducible expression of bcl-2 by the lac operator/repressor system in MDCK cells
Inducible expression of bcl-2 by the lac operator/repressor system in MDCK cells
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DOI：
10.1152/ajprenal.1997.273.2.f300
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发表时间：
1997-08-01
影响因子：
4.2
通讯作者：
Tang, MJ
中科院分区：
文献类型：
--
作者：
Lin, HH;Yang, TP;Tang, MJ
利用 lac 操纵子/阻遏物诱导系统来剖析人类 bcl-2 基因在 Madin-Darby 犬肾 (MDCK) 细胞中表达的生物学后果。将细胞转入组成型表达的 lad 基因（编码 lac 阻遏物）和 bcl-2 基因，该基因已插入包含 lac 操纵基因序列的猿猴病毒 40 (SV40) 启动子下游。因此，通过 lac 阻遏物与 SV40 调节区附近的 lac 操纵子序列结合，bcl-2 基因的表达可被抑制至基础水平，并可通过乳糖类似物异丙基-β-硫代半乳糖苷 (IPTG) 的施用而被特异性激活。我们发现0.01 mM IPTG可以诱导bcl-2基因的表达，并且在1 mM时获得最大诱导。经过IPTG处理，Bcl-2蛋白可在6小时内被诱导。此外，IPTG诱导的Bcl-2蛋白表达是一个可逆过程。最后，功能测定表明，IPTG 诱导的 bcl-2 基因表达分别赋予对无家可归细胞死亡或汇合细胞死亡的部分或完全抵抗。诱导表达系统对于剖析 bcl-2 对 MDCK 细胞表型或形态变化的影响特别有用。
The lac operator/repressor-inducible system was utilized to dissect the biological consequences of human bcl-2 gene expression in Madin-Darby canine kidney (MDCK) cells. Cells were made transgenic for a constitutively expressed lad gene, encoding lac repressor, and the bcl-2 gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of the bcl-2 gene could therefore be repressed to basal level by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region and be specifically activated by administration of the lactose analog isopropyl-beta-thiogalactoside (IPTG). We showed that expression of bcl-2 gene could be induced by 0.01 mM IPTG, and the maximal induction was obtained at 1 mM. With the treatment of IPTG, the Bcl-2 protein could be induced within 6 h. Moreover, the IPTG-inducible expression of Bcl-2 protein is a reversible process. Finally, functional assays revealed that IPTG-induced expression of bcl-2 gene conferred partial or complete resistance to homeless cell death or confluent cell death, respectively. The inducible expression system should be particularly useful for dissecting the effect of bcl-2 in phenotypic or morphological changes of MDCK cells.