Use of the human EF-1α promoter for expression can significantly increase success in establishing stable cell lines with consistent expression:: a study using the tetracycline-inducible system in human cancer cells
Use of the human EF-1α promoter for expression can significantly increase success in establishing stable cell lines with consistent expression:: a study using the tetracycline-inducible system in human cancer cells
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DOI：
10.1093/nar/27.24.4775
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发表时间：
1999-12-15
影响因子：
14.9
通讯作者：
Fisher, PB
中科院分区：
文献类型：
--
作者：
Gopalkrishnan, RV;Christiansen, KA;Fisher, PB
在四环素（Tc）的诱导控制下建立具有外源引入的目的基因的细胞最初需要稳定表达四环素激活剂（tTA或rtTA）的克隆细胞系。最初描述的表达tTA/rtTA的质粒载体由巨细胞病毒（CMV）立即早期（IE）启动子-增强子驱动，该启动子-增强子以其在广谱细胞类型中的稳健活性而闻名。虽然许多报道证明了这种构建体的实用性和功效，但遇到了无法建立具有所研究的cDNA的诱导表达的细胞系的情况。CMV启动子活性在细胞中的自发消退已被观察到在时间和细胞类型依赖性的方式。这可能是未能建立Tc应答细胞系的一个促成因素。我们在这里报告，改变表达盒的人延伸因子-1 α（EF-1 α）启动子已允许成功建立几个诱导型细胞系从不同的人肿瘤组织来源。我们解释这些结果意味着，灭绝的rtTA（或tTA）的表达可能是一个重要的因素，在建立TC诱导的细胞系缺乏成功。此外，本发现与需要使用稳定细胞系的实验具有普遍相关性。
Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1 alpha (EF-1 alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.