Departments of Medicine and Laboratory Medicine and Institute for Human Genetics, University of California, San Francisco, CA 94143, USA.
Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19467-72. doi: 10.1073/pnas.1012677107. Epub 2010 Oct 25.
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.
迄今为止，已有大量报告描述了使用不同数量的转录因子将多种体细胞类型重编程为诱导多能干细胞 (iPS) 细胞，并设计了将转录因子基因或蛋白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，并在非肥胖型糖尿病/严重免疫缺陷小鼠中形成了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，它们与相邻基因的距离从几百到 >100 万 bp 不等。可以通过 RT-PCR 研究插入对这些基因表达的影响。未检测到插入到 microRNA 基因座。因此，可以选择相邻基因功能不受影响的细胞，或者在必要时可以去除插入物。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内源性基因功能未受干扰的 iPS 细胞，并且可能对未来的人类治疗应用是安全的。
