Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis

Pontes, Samuel; Heredia, Luís; Casquero-García, Verónica; Fernández-Chacón, Macarena; Luo, Wen; Hermoso, Ana; Bansal, Mayank; García-González, Irene; Sanchez-Muñoz, Maria S.; Perea, Juan Ramón; Galiana-Simal, Adrián; Rodriguez‐Arabaolaza, Iker; Olmo-Cabrera, Sergio Del; Rocha, Susana; Criado-Rodríguez, Luis Miguel; Giovinazzo, Giovanna; Benedito, Rui

doi:10.1016/j.cell.2017.07.031

Cited by 76 publications

(99 citation statements)

References 35 publications

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“…More precise estimates of the number of medial SMCs that goes into forming the plaque SMC population in different atherosclerosis models and at different time points could be obtained using mosaic mice with more recombination outcomes than the hemizygous Confetti mice used by Chappell et al and us. Such tools have recently been published (23).…”

Section: Clonality and Phenotype Of Non-cap Smcsmentioning

confidence: 99%

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

et al. 2017

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Section: Clonality and Phenotype Of Non-cap Smcsmentioning

confidence: 99%

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

et al. 2017

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“…This enabled quantification of the contributions of multinucleate versus mononucleate cells to heart regeneration in mosaic animals (Gonzalez-Rosa et al, 2018). Similar applications in regeneration contexts can incorporate overexpressed molecular factors into Brainbow expression cassettes as shown recently in mice (Pontes-Quero et al, 2017) (Figure 3A), allowing association of a color with a high-resolution cellular phenotype caused by overexpression within a mosaic cell population.…”

Section: Finding Triggers and Brakes For Regenerationmentioning

confidence: 86%

“…Image in (A) is adapted from Pontes-Quero et al, 2017. Images in (B) are adapted from Kang et al, 2016.…”

Section: Figurementioning

confidence: 99%

A Regeneration Toolkit

Mokalled

Poss

2018

Developmental Cell

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Summary The ability of animals to replace injured body parts has been a subject of fascination for centuries. The emerging importance of regenerative medicine has reinvigorated investigations of innate tissue regeneration, and the development of powerful genetic tools has fueled discoveries into how tissue regeneration occurs. Here we present an overview of the armamentarium employed to probe regeneration in vertebrates, highlighting areas where further methodology advancement will deepen mechanistic findings.

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“…In particular, somatic transfection with iOn vectors provides a very efficient mean to track cell lineage in any organ accessible to electroporation, making them an ideal tool for studies in non-genetically tractable models. iOn also opens a simple route for functional mosaic analysis in vertebrate animals, normally requiring complex, time-consuming genetic manipulations (Pontes-Quero et al, 2017;Zong et al, 2005). In such experiments, the coupling of transgene integration and expression achieved with iOn ensures that marker expression in a cell reflects that of its entire lineage.…”

Section: Discussionmentioning

confidence: 99%

“…In vivo, transient episomal expression may confound the interpretation of somatic transgenesis experiments, because the past history of transgene expression in a population of cells may not be accurately reflected by the markers they harbor: as episomal transgenes are progressively lost through cell division, 2 they may alter the behavior of progenitors while being undetectable in their descent. This essentially precludes the development of reliable functional mosaic analysis schemes based on exogenous DNA vectors applicable to probe neural stem cell regulation, until now only possible by resorting to complex genetic schemes (Pontes-Quero et al, 2017;Zong et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Direct readout of neural stem cell transgenesis with an integration-coupled gene expression switch

Kumamoto

Maurinot

Barry‐Martinet

et al. 2019

Preprint

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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%...

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Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis

Cited by 76 publications

References 35 publications

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

A Regeneration Toolkit

Direct readout of neural stem cell transgenesis with an integration-coupled gene expression switch

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