Identification of genes associated with cortical malformation using a transposon-mediated somatic mutagenesis screen in mice

Lu, I-Ling; Chen, Chien; Tung, Chiou-Kou; Chen, Hsin‐Hung; Pan, Jiaping; Chang, Cheng-Chung; Cheng, Jia-Shing; Chen, Yi‐An; Wang, Chun‐Hung; Huang, Chia‐Wei; Kang, Yi-Ning; Chang, Hou‐Tai; Li, Lei-Li; Chang, Kuo-Ting; Shih, Yang-Hsin; Lin, Chi‐Hung; Kwan, Shang‐Yeong; Tsai, Jin Wu

doi:10.1038/s41467-018-04880-8

Cited by 28 publications

(32 citation statements)

References 65 publications

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“…In addition, a recent transposon-mediated mutagenesis screen using in utero electroporation of mouse embryos to detect mutant neural progenitor (radial glia) cells unable to migrate to the cortex identified 33 candidate genes for malformations of cortical development, one of which was Macf1. 20 Defects in axonal extension and guidance have also been seen with mutations in Short stop (shot), the Drosphila MACF1 homolog that interacts with Robo and Slit and regulates midline axon crossing. 21 In shot mutants, axonal growth cones initiate at the normal time and have normal morphology, including normal axon orientation and fasciculation.…”

Section: Mouse Knockoutsmentioning

confidence: 99%

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

Dobyns

Aldinger²,

Ishak

et al. 2018

The American Journal of Human Genetics

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To date, mutations in 15 actin-or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts.Microtubules (MTs) and filamentous actin form key structural components of the cytoskeleton, a dynamic intracellular structure that is essential for several basic cell functions, including migration, the formation of cellular processes (including axons and dendrites), axonal guidance, and vesicular trafficking. Mammalian genomes contain two spectraplakins-MACF1 (also known as ACF7) and DST (also known as BPAG1)-that function as actin-MT cross-linkers and essential integrators and modulators of cytoskeletal processes. [1][2][3] MACF1 is a large gene that expresses many isoforms, several of which are brain specific, through alternative splicing. The N terminus of the major isoforms contains two calponin-homology (CH) domains that bind actin, a plakin domain, and a long spectrin-repeat rod domain that confers flexibility. The C terminus of all isoforms functions as a MT binding domain and contains two calcium-binding EF-hand domains, a zinc-binding GAR (growth-arrest specific 2 or Gas2-related) domain, a positively charged Gly-Ser-Arg (GSR) region, and an EB1-binding SxIP domain. 1-3

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Section: Mouse Knockoutsmentioning

confidence: 99%

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

Dobyns

Aldinger²,

Ishak

et al. 2018

The American Journal of Human Genetics

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“…These experiments revealed that the PB-based IUE method provides a valuable new tool for tracking and manipulating neural lineages. Recently, Lu et al [39] applied IUE coupled with PB-mediated somatic mutagenesis to identify potential genes involved in the behavior of cortical neurons in the developing neocortex that often lead to malformations of cortical development (MCDs). IUE was performed at Day 14.5 of the embryonic stage, at the time when cortical neurogenesis is known to be most active.…”

Section: In Utero Gene Deliverymentioning

confidence: 99%

“…The maximum cargo size of viral vectors is restricted, hampering gene delivery of larger genes. Furthermore, construction of viral vectors is time-consuming, costly, and requires living cells for their large-scale production, which is also gene-wide screening of genes related to neural development and disorders [39]. PB is also known for its importance in inducing immortalization of cultured human deciduous tooth dental pulp cells [40] and hepatocytes in vivo [41].…”

Section: Introductionmentioning

confidence: 99%

“…Thus, DNA transposons, as exemplified by PB, are considered to be promising non-viral alternatives. For example, PB has been employed for a variety of applications, such as in vitro transfection in various mammalian cells, including human [23,24], bovine [25], goat [26], pig [27,28], and mice [14][15][16]29,30]; generation of genome-edited [31] and inducible pluripotent stem (iPS) cells [32][33][34]; generation of transgenic (Tg) animals [35][36][37][38]; and gene discovery via insertional mutagenesis through in vivo gene-wide screening of genes related to neural development and disorders [39]. PB is also known for its importance in inducing immortalization of cultured human deciduous tooth dental pulp cells [40] and hepatocytes in vivo [41].…”

Section: Introductionmentioning

confidence: 99%

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piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

et al. 2020

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In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. piggyBac (PB), one of three transposons (PB, Sleeping Beauty (SB), and Tol2) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.

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“…Seminal studies have taken advantage of retroviral vectors to characterize the neuronal and glial descent of embryonic neural progenitors in model animals (Noctor et al, 2001;Price et al, 1987;Yu et al, 2012). The location of these progenitors along the lumen of the neural tube also makes them accessible to integrative schemes based on DNA electroporation applicable to trace neural cell lineage (Chen and Loturco, 2012;Loulier et al, 2014), target specific genes and mark their product (Mikuni et al, 2016;Suzuki et al, 2016), or even perform in situ screens for genes involved in neurodevelopmental processes (Lu et al, 2018).…”

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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Identification of genes associated with cortical malformation using a transposon-mediated somatic mutagenesis screen in mice

Cited by 28 publications

References 65 publications

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

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

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