Quantitative comparison of constitutive promoters in human ES cells.
Quantitative comparison of constitutive promoters in human ES cells.
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DOI：
10.1371/journal.pone.0012413
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发表时间：
2010-08-26
期刊：
影响因子：
3.7
通讯作者：
Semb H
中科院分区：
文献类型：
--
作者：
Norrman K;Fischer Y;Bonnamy B;Wolfhagen Sand F;Ravassard P;Semb H
组成启动子是一种确保基因持续高水平表达的基础研究工具，具有广泛的应用，包括人类胚胎学研究和人类胚胎干细胞（hESCs）的药物发现。许多细胞/病毒启动子确保在各种细胞类型中持续的基因表达已经被确定，但它们在hESCs中的活性仍然缺乏系统的比较。我们定量比较了五种常用的组成型启动子的启动子活性，包括人β-肌动蛋白启动子（ACTB）、巨细胞病毒（CMV）、延伸因子-1α (EF1α)、磷酸甘油酸激酶（PGK）和泛素（UbC）。使用慢病毒基因转移确保启动子egfp构建物稳定整合到hESCs基因组中。在未分化hESCs及其分化后代的长期培养中定量比较启动子活性。ACTB、EF1α和PGK启动子在未分化hESCs的长期培养中表现出稳定的活性。ACTB启动子在培养50天后仍能在75-80%的细胞中保持表达。胚状体（EB）分化过程中，5个启动子活性均降低。虽然EF1α启动子在大约50%的细胞中下调，但它是分化过程中最稳定的启动子。分化的eGFP+和eGFP-细胞的基因表达分析表明，启动子活性可能仅限于特定的细胞系，这表明需要在分化的hESCs中仔细选择最佳的组成基因表达启动子。
Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75–80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.
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