El-Mogy Mohamed A, Abdalla Moemen A K, Misic Vanja, Haj-Ahmad Yousef
Molecular Biology Department, National Research Centre, Dokki, Giza, Egypt.
Department of Biological Sciences, Brock University, St. Catharines, ON, Canada.
J Genet Eng Biotechnol. 2017 Jun;15(1):211-217. doi: 10.1016/j.jgeb.2017.04.003. Epub 2017 Apr 21.
The adenovirus major late promoter (MLP) and its translational regulator - the tripartite leader (TPL) sequence - can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie) promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO) cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds) than translation (11 folds). Even with adenovirus infection to provide additional elements (), CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.
腺病毒主要晚期启动子（MLP）及其翻译调节因子——三联体前导序列（TPL）——在腺病毒感染期间可有效驱动高效基因表达。然而，在腺病毒基因组背景之外的转基因表达中，这两个元件尚未得到广泛测试。在本研究中，我们测试了MLP和TPL的组合是否能在哺乳动物细胞转基因表达中，使转基因表达水平超过最广泛使用的启动子——巨细胞病毒立即早期（CMVie）启动子/增强子。在中国仓鼠卵巢（CHO）细胞中比较了这两个调控元件的活性。尽管与MLP/TPL相比，CMVie启动子/增强子控制下的瞬时表达显著更高，但转录水平的差异（30倍）大于翻译水平的差异（11倍）。即使通过腺病毒感染提供额外元件，CMVie启动子/增强子相对于MLP/TPL仍表现出显著更高的活性。有趣的是，CMVie启动子/增强子在腺病毒感染细胞中的活性比未感染细胞高1.9倍。我们的研究表明，MLP-TPL驱动的转基因表达低于CMVie启动子/增强子，尤其是在转录水平。数据还突出了TPL序列在翻译水平的作用，以及CMVie启动子/增强子的高转录活性可能使细胞翻译机制不堪重负。此外，我们在此展示的数据表明腺病毒感染可刺激CMVie启动子/增强子，这在未来测试CMVie/TPL序列与其他腺病毒元件组合用于转基因表达的研究中可能会很有趣。
