As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We ...have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.
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•Rapid brain tumor modeling with postnatal electroporation and transposon methodology•Modeling methodology allows for extensive interrogation of tumor growth mechanisms•Ras pathway mutations deplete neural stem cells and upregulate Ets factors•Ets signaling block rescues Ras-mediated stem cell loss and prevents tumor formation
Breunig et al. report that increased Ras signaling functions to deplete neural stem cells and expand glial progenitors in gliomagenesis. Inhibition of the upregulated Ets signaling downstream of Ras is sufficient to inhibit glioma formation by attenuating the gliogenesis necessary for tumor propagation.
Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that ...CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (RhoS334) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of RhoS334, which prevented retinal degeneration and improved visual function.
The generation of neurons and glia from radial glia progenitors is critical to proper neocortical development but the mechanisms regulating their deterministic production are unclear. In this issue ...of Neuron, Beattie et al. (2017) use elegant MADM-based lineage tracing to demonstrate cell-intrinsic and global functions for Lgl1 during neocortical development.
The generation of neurons and glia from radial glia progenitors is critical to proper neocortical development but the mechanisms regulating their deterministic production are unclear. In this issue of Neuron, Beattie et al. (2017) use elegant MADM-based lineage tracing to demonstrate cell-intrinsic and global functions for Lgl1 during neocortical development.
Precise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We ...engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants—along with engineered mutants—in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.
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•pB-Tet-GOI features the latest generation of Tet transactivators (tTAs) and variants•piggyBac transposition allows for genomic insertion of pb-Tet-GOI•pb-Tet-GOI provides flexible control of transgenes in vitro and in vivo•tTA variants permit reversible, constitutive, or induced constitutive expression
In this article, Breunig and colleagues describe a stable, flexible genetic system for inducible and reversible gene expression. The doxycycline-mediated system, termed pB-Tet-GOI, is non-leaky, robust, and allows for “On→Off→On,” “Off→On→Off” and inducible-constitutive temporal control (“Off→permanently On”) of transgenes in vivo and in vitro, as well as a dox-resistant variant for strong, constitutive expression of transgenes.
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish ...mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with “personalized” driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice.
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•MADR enables single-locus somatic genetic modification and lineage tracing in vivo•MADR can be used to interrogate the dosage response of transgenes in vivo•MADR allows “personalized” brain tumor modeling in vivo via CRISPR variants or transgenes•MADR is adaptable to hundreds of available mouse lines
Mosaic analysis with dual recombinase-mediated cassette exchange (MADR) is a simple, fast, and generalizable method for the generation of stable, defined copy number somatic transgenic animals that can accelerate research investigations of development and disease.
Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a ...promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.
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•Created plasmid with tetracycline transactivator along with dual reporters and GDNF•Efficient, stable transduction of human iPSC-derived neural progenitor cells•Inducible and reversible in vivo expression of GDNF, reporter protein, and luciferase•Promising stem cell and gene therapy strategy for neurodegenerative diseases
Svendsen, Breunig, and colleagues generated a plasmid containing a tetracycline transactivator and a response element expressing dual reporters and GDNF. Stably transduced human iPSC-derived neural progenitors show inducible, reversible expression of GDNF, a fluorescent reporter protein, and luciferase in vitro and following transplantation into the mouse brain. This combined cell and gene therapy advancement is promising for neurodegenerative diseases.
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