Varas Florencio, Stadtfeld Matthias, de Andres-Aguayo Luisa, Maherali Nimet, di Tullio Alessandro, Pantano Lorena, Notredame Cedric, Hochedlinger Konrad, Graf Thomas
Differentiation and Cancer, Center for Genomic Regulation and Pompeu Fabra University, Barcelona, Spain.
Stem Cells. 2009 Feb;27(2):300-6. doi: 10.1634/stemcells.2008-0696.
Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.
几个实验室报告称，利用携带Oct4、Sox2、Klf4和c-Myc转录因子基因的逆转录病毒，已将小鼠和人类成纤维细胞重编程为多能细胞。在这些实验中，重编程频率低于被感染细胞的0.1%，这增加了诱导重编程需要其他事件的可能性，比如由逆转录病毒插入触发的基因激活。因此，我们通过连接介导的聚合酶链反应（LM-PCR）确定了来自小鼠成纤维细胞的6个诱导多能干细胞（iPS）克隆中的逆转录病毒插入位点。79个插入位点被定位到单个小鼠基因组位置。其中35个映射到基因转录单元，而29个插入落在转录起始位点的10千碱基范围内。在所研究的iPS克隆中未检测到共同的插入位点。此外，生物信息学分析显示，在逆转录病毒插入靶向的基因中，没有特定基因功能、网络或途径的富集。我们得出结论，Oct4、Sox2、Klf4和c-Myc足以促进成纤维细胞到iPS细胞的重编程，并提出观察到的低重编程频率可能有其他解释。
