Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.
Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.
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DOI：
10.1038/nmeth.1323
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发表时间：
2009-05
期刊：
影响因子：
48
通讯作者：
Bradley, Allan
中科院分区：
文献类型：
--
作者：
Yusa, Kosuke;Rad, Roland;Takeda, Junji;Bradley, Allan
诱导多能干细胞 (iPSC) 是通过 Oct4、Sox2、Klf4 和 cMyc 的转基因表达从体细胞产生的。然而，将该技术应用于再生医学的一个主要困难是重编程因子的传递。尽管逆转录病毒转导会增加致瘤性的风险，但瞬时表达方法的重编程效率却相当低。在这里，我们展示了一种基于 PiggyBac 转座子的高效方法来生成免整合 iPSC。携带2A肽连接重编程因子的转座子诱导小鼠胚胎成纤维细胞重编程，其效率与逆转录病毒转导相当。通过重新表达转座酶从这些原代 iPSC 中去除转座子。通过 HSVtk-FIAU 选择可以轻松识别无转基因的 iPSC。 PiggyBac 进行无足迹切除，使 iPSC 基因组不发生任何遗传改变。 iPSC 满足多能性的所有标准，例如胚胎干细胞特异性标记物的表达、畸胎瘤的形成和对嵌合体的贡献。基于piggyBac转座子的重编程可用于产生治疗上适用的iPSC。
Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4, Sox2, Klf4, and cMyc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we show a highly efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. Transposons were removed from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be easily identified by HSVtk-FIAU selection. piggyBac excises without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as expression of embryonic stem cell-specific markers, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.
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DOI：
10.1126/science.1162494
发表时间：
2008-11-07
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Stadtfeld M;Nagaya M;Utikal J;Weir G;Hochedlinger K
通讯作者：
Hochedlinger K
影响因子：
8
作者：
Foster, KW;Liu, ZL;Ruppert, JM
通讯作者：
Ruppert, JM
影响因子：
64.5
作者：
Hanna, Jacob;Markoulaki, Styliani;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
5.3
作者：
Niwa, H;Masui, S;Miyazaki, J
通讯作者：
Miyazaki, J
影响因子：
64.5
作者：
Ding, S;Wu, XH;Xu, T
通讯作者：
Xu, T