Sorbonne Université, INSERM, CNRS, Institut de la Vision, 75012 Paris, France.
Institut du Fer à Moulin, 75005 Paris, France; INSERM, UMR-S 1270, 75005 Paris, France; Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France.
Neuron. 2020 Aug 19;107(4):617-630.e6. doi: 10.1016/j.neuron.2020.05.038. Epub 2020 Jun 18.
Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.
稳定的外源性转基因在神经发育和干细胞研究中至关重要。尽管 DNA 载体驱动的工具使基因组插入越来越高效，但转基因仍然受到无法区分整合和附加型转基因的根本限制。在这里，我们引入了一种整合偶联的 On 遗传开关，iOn，它通过转座将插入宿主基因组时触发基因表达，从而能够在转染裸质粒后快速准确地识别整合事件。在体外，iOn 允许使用多种载体快速、无药物稳定转化小鼠和人类多能干细胞。在体内，我们证明了在体细胞转基因实验中，细胞谱系追踪、调控元件评估和基因功能嵌合体分析的准确性，揭示了神经祖细胞的潜能和相互作用。这些结果确立了 iOn 作为一种通用策略的地位，通过将转基因激活条件与基因组整合，加速和简化培养系统和模式生物中的基因工程。
