Pertek Anna, Meier Florian, Irmler Martin, Beckers Johannes, Skylaki Stavroula, Endele Max, Wurst Wolfgang, Prakash Nilima, Kühn Ralf
Institute of Developmental Genetics, Helmholtz Zentrum München, 85764, Munich, Germany,
Mol Biotechnol. 2014 Aug;56(8):697-713. doi: 10.1007/s12033-014-9748-y.
Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.
哺乳动物细胞可被重编程为诱导多能干细胞（iPSC），这是一种用于体外疾病建模和再生医学的宝贵工具。这些应用需要不含重编程因子转基因的iPSC，但目前获得无转基因iPSC的方法效率低下且繁琐。在这里，我们描述了一种新方法，通过顺序使用两种DNA重组酶C31整合酶和Cre来控制单个非病毒重编程载体的基因组插入和切除，从而简单地获得无转基因iPSC。我们表明，这种无转基因iPSC表现出与真正的、囊胚来源的胚胎干细胞相当的基因表达谱和多能发育潜力。如一个用于分化为中脑多巴胺能神经元的报告iPSC系所示，双重组酶方法为获得用于研究疾病机制和细胞替代疗法的无转基因iPSC提供了一种简单有效的方法。
