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.
Cancer cell secretion of TGF-β is a potent mechanism for immune evasion. However, little is known about how central nervous system tumors guard against immune eradication. We sought to determine the ...impact of T-cell TGF-β signaling blockade on progression of medulloblastoma (MB), the most common pediatric brain tumor. Genetic abrogation of T-cell TGF-β signaling mitigated tumor progression in the smoothened A1 (SmoA1) transgenic MB mouse. T regulatory cells were nearly abolished and antitumor immunity was mediated by CD8 cytotoxic T lymphocytes. To define the CD8 T-cell subpopulation responsible, primed CD8 T cells were adoptively transferred into tumor-bearing immunocompromised SmoA1 recipients. This led to generation of CD8 ⁺/killer cell lectin-like receptor G1 high (KLRG1 ʰⁱ)/IL-7R ˡᵒ short-lived effector cells that expressed granzyme B at the tumor. These results identify a cellular immune mechanism whereby TGF-β signaling blockade licenses the T-cell repertoire to kill pediatric brain tumor cells.
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.
There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines ...transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres.
Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated.
Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled
C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS.
These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable metabolically by analyzing expression profiles and glucose consumption. Our results also highlight important differences in nucleotide synthesis utilization and DNA repair capacity that could be exploited for therapy. Altogether, this study demonstrates that IDH1 mutant gliomas are a distinct subclass of glioma with a less malignant, but also therapy-resistant, metabolic profile that will likely require distinct modes of therapy.
Recent findings have indicated the presence of a progenitor domain at the marginal zone/layer 1 of the cerebral cortex, and it has been suggested that these progenitors have neurogenic and gliogenic ...potential. However, their contribution to the histogenesis of the cortex remains poorly understood due to difficulties associated with genetically manipulating these unique cells in a population-specific manner.
We have adapted the electroporation technique to target pial surface cells for rapid genetic manipulation at postnatal day 2. In vivo data show that most of these cells proliferate and progressively differentiate into both neuronal and glial subtypes. Furthermore, these cells localize to the superficial layers of the optic tectum and cerebral cortex prior to migration away from the surface.
We provide a foundation upon which future studies can begin to elucidate the molecular controls governing neural progenitor fate, migration, differentiation, and contribution to cortical and tectal histogenesis. Furthermore, specific genetic targeting of such neural progenitor populations will likely be of future clinical interest.
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.
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