Summary 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.

Stable genomic integration of exogenous transgenes is critical for neurodevelopmental and neural stem cell studies. Despite the emergence of tools driving genomic insertion at high rates with DNA vectors, transgenesis procedures remain fundamentally hindered by the impossibility to distinguish integrated transgenes from residual episomes. Here, we introduce a novel genetic switch termed iOn that triggers gene expression upon insertion in the host genome, enabling simple, rapid and faithful identification of integration events following transfection with naked plasmids accepting large cargoes. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate accurate cell lineage tracing, assessment of regulatory elements and mosaic analysis of gene function in somatic transgenesis experiments that reveal new aspects of neural progenitor potentialities and interactions. These results establish iOn as an efficient and widely applicable strategy to report transgenesis and accelerate genetic engineering in cultured systems and model organisms. foundations to T. K., the French ministry of research to F.M. and C.V., by the European Research Council (ERC-CoG n° 649117), and by Agence Nationale de la Recherche (contracts ANR-10-LABX-65, ANR-10-LABX-73-01 and ANR-15-CE13-0010-02). S.N. was funded by ATIP/Avenir and Association Française contre les Myopathies and A.R. by Genespoir. AUTHOR CONTRIBUTIONS J.L., R.B. and T.K. conceived the iOn and LiOn switches. Initial experiments, R.B.; in vitro and chick spinal cord experiments, T.K.; retina experiments, F.M. and A.R.; cortical electroporation, T.K. and K.L.; iPS cell experiments, C.V. and S.N.; ES cell experiments, M.C.T. and S.V.-P.; cell 9 100 ng iOn vector with or without 20 ng of PBase-expressing plasmid (CAG::hyPBase) using 0.7 µl of Lipofectamine 2000 reagent (Invitrogen). For triple-color labeling experiments, we used 100 ng/well of each LiOn CAG∞FP plasmid and 60 ng of PBase vector. To validate the LiOn CMV∞Cre transgene, 50 ng of the corresponding plasmid was co-transfected with 10 ng of PBase vector in a HEK293 cell line stably expressing the Tol2 CAG::RY reporter. This line was established by successive use of Tol2 transposition, drug selection with G418 (300 μg/ml, Sigma) and picking of RFP-positive clones. In some experiments, 50 ng of non-integrative plasmid expressing an FP marker distinct from the iOn vector (CMV::mTurquoise2, CMV::IRFP or CAG::GFP) were applied as transfection control. For FACS analysis, transfections were performed in 6-cm dishes with scaled up concentrations. HEK293 cell viability after iOn plasmids transfection was assessed by dye exclusion with Trypan blue solution (0.4%, Sigma).FP expression was either assayed by flow cytometry, epifluorescence or confocal microscopy, or an Arrayscan high-content system (Thermo Fisher Scientific) (see below). For fixed observations, cells grown on 13 mm coverslips coated with collagen (50 μg/ml, Sigma) were immersed in 4%...