Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP⁺ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34⁺ CD45⁺ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

DNA甲基化在哺乳动物细胞中短暂治疗基因的表达是基因治疗中的主要挑战之一。在这项研究中，我们评估了DNA甲基化对小鼠胚胎干（ES）和小鼠诱导性多能干（iPS）细胞中GFP表达持续时间的影响。用携带绿色荧光蛋白（GFP）的慢病毒（LV）转导细胞，慢病毒（LV）由人延伸因子（EF1α）或巨细胞病毒（CMV）启动子驱动。转导的iPS细胞表现出更高的GFP ⁺百分比具有比转导ES细胞持久的平均荧光强度的细胞。对转基因细胞群体中转基因整合拷贝的分析显示出相似的拷贝数。然而，仅在转导的ES细胞中观察到5-azaC处理后GFP强度的显着增加，表明DNA甲基化对转基因沉默的影响。随后的DNA甲基化分析表明，与转导的ES细胞相比，iPS细胞中原病毒的启动子和GFP区域的甲基化分布可忽略不计。有趣的是，在转导的iPS细胞向CD34 ⁺ CD45 ⁺定向分化后观察到持续的转基因表达细胞。因此，这项研究表明，慢病毒转导的iPS细胞具有良好的转基因活性是由于前病毒区缺乏甲基化。