Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors
Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors
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DOI：
10.1073/pnas.0806976105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Mitani, Kohnosuke
中科院分区：
文献类型：
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作者：
Suzuki, Keiichiro;Mitsui, Kaoru;Mitani, Kohnosuke
人类胚胎干细胞（hES）被认为是再生医学细胞材料的潜在无限来源。对于使用灵长类胚胎干细胞的生物学研究和临床应用，开发一种通用策略来获得有效的基因传递和基因操作，特别是通过同源重组（HR）进行基因靶向，将是至关重要的。然而，与小鼠ES （mES）细胞不同，hES细胞中瞬时基因传递和HR的有效策略尚未建立。在这里，我们报道了辅助依赖腺病毒载体（HDAdVs）能够有效地在hES和食蟹猴（Macaca fasicularis） ES （cES）细胞中转移基因。在不失去胚胎干细胞未分化状态的情况下，瞬时基因转移效率接近100%。使用带有同源臂的hdadv，大约十分之一的载体染色体整合是通过HR进行的，而使用其他基因传递方法，这一比例仅接近1%。此外，结合负选择，大约45%的载体染色体整合是靶向整合，这表明HDAdVs将成为在hES细胞和其他类型的人类干细胞（如诱导多能干细胞（iPS）细胞）中进行遗传操作的强大工具。
Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was approximate to 100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only approximate to 1% with other gene delivery methods. Furthermore, in combination with negative selection, approximate to 45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.