Promoter-dependent enchanced green fluorescent protein expression during embryonic stem cell propagation and differentiation
Promoter-dependent enchanced green fluorescent protein expression during embryonic stem cell propagation and differentiation
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胚胎干细胞增殖和分化过程中启动子依赖性增强的绿色荧光蛋白表达
DOI：
10.1089/scd.2007.0084
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发表时间：
2008-04-01
影响因子：
4
通讯作者：
Yang, Huang-Tian
中科院分区：
文献类型：
--
作者：
Wang, Rong;Liang, Ji;Yang, Huang-Tian
遗传修饰是胚胎干细胞研究的重要工具，需要高效的启动子系统。在这里，我们比较了三种普遍存在的启动子，延伸因子-1 α（EF 1 α），磷酸甘油酸激酶-1（PGK），和巨细胞病毒（CMV），在小鼠（m）ES细胞的繁殖和分化过程中的转录活性，通过使用稳定的mES细胞系表达增强的绿色荧光蛋白（EGFP）下的每一个这些启动子。在未分化的ES细胞中，由EF 1 α驱动的EGFP表达最稳定，其次是PGK，而由CMV启动子驱动的EGFP表达的下调在直至第35代的繁殖期间最显著。在14天的分化过程中，在胚状体（EB）中观察到这些启动子的活性的类似模式，与其他两个相比，EF 1 α启动子驱动的EGFP信号更亮。此外，EF 1 α和PGK启动子，而不是CMV，在几乎所有的mES细胞分化的神经元细胞，心肌细胞，内脏内胚层细胞中是有效的，与荧光信号强度较高的EF 1 α，甚至PGK。CMV启动子在约60-80%的这些分化细胞中产生弱荧光信号，而少数具有CMV启动子的分化细胞显示出与具有EF 1 α启动子的细胞一样的明亮的EGFP表达。这些结果扩展了先前对这些启动子在mES细胞中的活性的观察，并为选择合适的启动子以促进mES细胞的遗传修饰提供了新的信息。
Genetic modification is an important tool in embryonic stem (ES) cell research and requires efficient promoter systems. Here, we have compared the transcriptional activities of three ubiquitous promoters, elongation factor-1 alpha (EF1 alpha), phosphoglycerate kinase-1 (PGK), and cytomegalovirus (CMV), during propagation and differentiation of mouse (m) ES cells by using stable mES cell lines expressing enhanced green fluorescent protein (EGFP) under each of these promoters. In undifferentiated ES cells, the EGFP expression driven by the EF1 alpha was most stable, followed by the PGK, whereas the down-regulation of EGFP expression driven by the CMV promoter was most significant during propagation up to passage 35. A similar pattern for the activities of these promoters was observed in embryoid bodies (EBs) during 14 days of differentiation, with brighter EGFP signals driven by the EF1 alpha promoter versus the other two. Moreover, the EF1 alpha and PGK promoters, but not CMV, were effective in almost all mES cell-differentiated neuronal cells, cardiomyocytes, and visceral endoderm cells, with the fluorescent signal intensity higher for EF1 alpha and even for PGK. The CMV promoter yielded a weak fluorescent signal in about 60-80% of these differentiated cells, while a few differentiated cells with the CMV promoter showed bright EGFP expression like that with the EF1 alpha promoter. These results extend previous observations for the activities of these promoters in mES cells and provide new information for choosing appropriate promoters to facilitate genetic modification of mES cells.