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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Generation of induced pluripotent stem cells using site-specific integration with phage integrase
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Generation of induced pluripotent stem cells using site-specific integration with phage integrase

机译：使用噬菌体整合酶的位点特异性整合产生诱导性多能干细胞

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To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and- formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.
机译：迄今为止，大量报告描述了使用不同数量的转录因子将许多体细胞类型重编程为诱导多能干（iPS）细胞，并设计了将转录因子基因或蛋白质引入体细胞的替代方法。在这里，我们描述了一种使用噬菌体ΦC31整合酶将小鼠胚胎成纤维细胞和人羊水细胞重编程为iPS细胞的方法。这些iPS细胞在非肥胖/严重受损的免疫缺陷小鼠中表现出与ES细胞相似的形态，表面抗原，基因表达和表观遗传状态，并形成具有三个胚层的畸胎瘤。重要的是，这些iPS细胞在每个细胞系中只有一个整合位点。整合的位置有利于基因间区域，并且它们与相邻基因的距离从几百个bp扩展到> 1百万bp。插入对这些基因表达的影响可以通过RT-PCR研究。没有检测到microRNA基因位点的插入。因此，可以选择不影响相邻基因功能的细胞，或者在必要时可以去除插入片段。我们得出结论，噬菌体整合酶介导的位点特异性重组可以产生具有不受干扰的内源基因功能的iPS细胞，并且对于将来的人类治疗应用可能是安全的。

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