Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo
Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo
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DOI：
10.1038/mt.2012.46
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发表时间：
2012-05-01
影响因子：
12.4
通讯作者：
Glimm, Hanno
中科院分区：
文献类型：
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作者：
Herbst, Friederike;Ball, Claudia R.;Glimm, Hanno
慢病毒载体（LV）广泛用于将基因稳定地转移到靶细胞中以研究或治疗基因功能。此外，可以改进向早期鼠胚胎中的基因转移以有效地产生转基因小鼠。我们应用慢病毒基因转移来产生SET结合蛋白1（Setbp 1）和增强型绿色荧光蛋白（eGFP）转基因小鼠模型。转基因创始人和他们的载体阳性后代都没有转录或表达转基因。内脾病灶形成病毒（SFFV）启动子的亚硫酸氢盐测序证明了转基因小鼠中所有分析的CpG的广泛甲基化。为了分析Setbp 1对表观遗传沉默的影响，在体外将胚胎干细胞（ESC）分化为心肌细胞（CM）。与LV中的人启动子相反，病毒衍生的启动子序列在分化期间强烈甲基化，独立于转基因。此外，常用的SFFV启动子（SFFV β）是高度甲基化的显着的强度和频率在造血分化过程中在体内LV，但较少在γ-逆转录病毒（γ-RV）的骨干。总之，我们得出结论，LV使用内部SFFVp是不适合产生转基因小鼠或在分化细胞中进行组成型表达研究。选择合适的启动子也是临床基因治疗中允许稳定的转基因表达的关键。
Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in gamma-retroviral (gamma-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.