Efficient exogenous DNA-free reprogramming with suicide gene vectors
Efficient exogenous DNA-free reprogramming with suicide gene vectors
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DOI：
10.1038/s12276-019-0282-7
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发表时间：
2019-07-19
影响因子：
12.8
通讯作者：
Kim, Janghwan
中科院分区：
文献类型：
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作者：
Lee, Minhyung;Ha, Jeongmin;Kim, Janghwan
使用附加型载体进行重编程是一种简单、安全且经济高效的方法，可产生无外源 DNA 的诱导多能干细胞 (iPSC)。然而，偶尔观察到外源基因的基因组整合。此外，在已建立的 iPSC 中，游离 DNA 的去除需要 70 多天的时间。在这里，我们将来自酵母的胞嘧啶脱氨酶 (CD) 基因插入附加型载体，并用它们将人类成纤维细胞重编程为 iPSC。这些新的附加型载体（CD 附加型载体）早在 5-氟胞嘧啶 (5-FC) 处理后 7 天就从生成的 iPSC 中消除。我们还发现，整合了 CD 基因的细胞在 5-FC 处理的两天内死亡。此外，我们通过使用 CD 附加型载体直接重编程并结合 5-FC 处理，在传代后产生了无外源的诱导神经干细胞。总之，我们的新方法可以快速、轻松地分离无外源重编程细胞，并可应用于疾病建模和临床应用。
Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.