Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner
Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner
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DOI：
10.1089/scd.2006.0057
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发表时间：
2007-02-01
影响因子：
4
通讯作者：
Zhang, Su-Chun
中科院分区：
文献类型：
--
作者：
Xia, Xiaofeng;Zhang, Yingsha;Zhang, Su-Chun
慢病毒越来越多地被用于包括胚胎干细胞在内的人类细胞的基因修饰。在慢病毒载体中利用四种普遍存在的启动子--巨细胞病毒(CMV)、巨细胞病毒即刻早期增强子/鸡β-肌动蛋白杂交体(CAG)、磷酸甘油酸激酶(PGK)和人延长因子-1α(EF1α)--驱动增强型绿色荧光蛋白(EGFP)基因在人ES细胞和小鼠ES细胞中的表达，通过评估转导了EGFP基因但未发出绿色荧光的细胞的百分比来确定EGFP抑制的程度。与小鼠ES细胞相比，在人ES细胞中观察到了更高水平的转基因抑制。这种抑制也高度依赖于启动子，导致在CMV或CAG启动子下超过95%的EGFP基因失活，而在PGK启动子下只有55%。瞬时转染人ES细胞未观察到启动子依赖的抑制作用。因此，在人类ES细胞中普遍存在的转基因表达低下的现象可能主要是由于转基因和整合后对转基因的抑制所致。慢病毒用于人ES细胞的基因修饰时，应注意选择最佳启动子。
Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters-cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken beta-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1 alpha (EF1 alpha) - in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.