The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

译文

人类胚胎干 (hES) 细胞系的可用性稳步增长,引起了人们对将鼠细胞中的基因转移工具应用于人类系统的浓厚兴趣。在这里,我们提出了一种用生态逆转录病毒载体转导hES细胞的方法。用携带鼠逆转录病毒受体mcat1的构建体瞬时转染hES细胞。随后,将细胞暴露于复制缺陷型莫洛尼鼠白血病病毒 (momoulv) 衍生物或假型慢病毒载体。对于oncoretrovirus载体,该程序产生高达20% 的总体转导效率,并允许选择具有高频率的永久转导克隆。选定的克隆保持了多能性相关标记的表达,并在体外和体内均表现出多胚层分化。HES细胞衍生的体细胞 (包括神经后代) 保持高水平的转基因表达。用moulv包膜假型的慢病毒载体可以以相同的方式引入,效率高达33%。即使没有选择压力,慢病毒转导的hES细胞的转基因表达在分化后仍保持永久性。该方法绕过了与使用两性逆转录病毒系统相关的调节问题,并利用了现有的大量鼠载体,为hES细胞及其后代中的基因转移和谱系分析提供了一种安全且通用的工具。

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