Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies - A Cautionary Reminder
Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies - A Cautionary Reminder
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DOI：
10.1055/s-2008-1078717
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发表时间：
2008-11-01
影响因子：
2.2
通讯作者：
Ludgate, M.
中科院分区：
文献类型：
--
作者：
Fuhrer, D.;Han, S.;Ludgate, M.
报告基因结构在确定真核生物基因调控和表达方面有着广泛的应用。体外模型经常需要共转染，并且有报道称启动子之间存在干扰，主要是抑制。研究促甲状腺素受体突变体的生物活性涉及受体构建体和由糖蛋白激素α亚基启动子驱动的cAMP响应荧光素酶报告基因的共转染。我们观察到，通过共转染含有SV40晚期启动子的空表达载体，基础荧光素酶活性显著增强。我们研究了在短暂共转染后，在COS细胞中使用不同浓度和几种病毒启动子的机制。这种增加是剂量依赖性的，但在50ng载体DNA时趋于稳定。这不是由于佐剂效应，因为添加越来越多的无启动子质粒并没有改变荧光素酶的活性。当将启动子截断至-346时，增强作用得以维持，但在-244上游的启动子中减弱，这表明在-244和-346之间存在假定的糖蛋白激素α表达抑制因子的结合位点。通过添加第二个组成报告基因（bos β -gal）来纠正转染效率，增强得以维持，尽管这是bos β -gal增强荧光素酶活性的结果，其本身被SV40启动子抑制。报告者活动的人为增强可能发生，并强调在进行瞬时共转染实验时需要仔细选择对照。启动子的计算机分析确定了一个可能的共享叉头转录因子结合位点。
Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. in vitro models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha Subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.