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1.
Cell ; 179(1): 251-267.e24, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31539496

RESUMO

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. VIDEO ABSTRACT.


Assuntos
Neoplasias Encefálicas/genética , Modelos Animais de Doenças , Marcação de Genes/métodos , Loci Gênicos/genética , Glioma/genética , Mutagênese Insercional/métodos , Transgenes/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Recombinases/metabolismo , Transfecção
2.
Mol Ther ; 24(3): 556-63, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26666451

RESUMO

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 (Rho(S334)) 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 Rho(S334), which prevented retinal degeneration and improved visual function.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Proteínas rho de Ligação ao GTP/genética , Alelos , Animais , Sítios de Ligação , Ordem dos Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Mutação , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , RNA Guia de Cinetoplastídeos , Ratos , Distrofias Retinianas/genética , Distrofias Retinianas/patologia , Distrofias Retinianas/terapia , Retinose Pigmentar/terapia , Sinapses/metabolismo
3.
Proc Natl Acad Sci U S A ; 111(33): E3458-66, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25082897

RESUMO

Cancer cell secretion of TGF-ß 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-ß signaling blockade on progression of medulloblastoma (MB), the most common pediatric brain tumor. Genetic abrogation of T-cell TGF-ß 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-ß signaling blockade licenses the T-cell repertoire to kill pediatric brain tumor cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Comportamento Animal , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL
4.
Cancer Metab ; 6: 4, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29692895

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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 13C-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. CONCLUSIONS: 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.

5.
Stem Cell Reports ; 4(3): 323-31, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25702640

RESUMO

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.


Assuntos
Linhagem da Célula/genética , Elementos de DNA Transponíveis , Expressão Gênica , Vetores Genéticos/genética , Células-Tronco/metabolismo , Transgenes , Animais , Linhagem Celular , Regulação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Humanos , Camundongos , Plasmídeos/genética
6.
Cell Rep ; 12(2): 258-71, 2015 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-26146073

RESUMO

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.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas ras/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Glioma/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurofibromina 1/antagonistas & inibidores , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para Cima
7.
J Vis Exp ; (87)2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24836046

RESUMO

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.


Assuntos
Eletroporação/métodos , Pia-Máter/fisiologia , Animais , Linhagem da Célula , Eletroporação/instrumentação , Camundongos , Pia-Máter/citologia
8.
Mol Neurobiol ; 45(3): 564-70, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22644387

RESUMO

Although the discovery of cilia is one of the earliest in cell biology, the past two decades have witnessed an explosion of new insight into these enigmatic organelles. While long believed to be vestigial, cilia have recently moved into the spotlight as key players in multiple cellular processes, including brain development and homeostasis. This review focuses on the rapidly expanding basic biology of neural cilia, with special emphasis on the newly emerging B9 family of proteins. In particular, recent findings have identified a critical role for the B9 complex in a network of protein interactions that take place at the ciliary transition zone (TZ). We describe the essential role of these protein complexes in signaling cascades that require primary (nonmotile) cilia, including the sonic hedgehog pathway. Loss or dysfunction of ciliary trafficking and TZ function are linked to a number of neurologic diseases, which we propose to classify as neural ciliopathies. When taken together, the studies reviewed herein point to critical roles played by neural cilia, both in normal physiology and in disease.


Assuntos
Cílios/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Organogênese , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Cílios/patologia , Humanos , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Neurônios/patologia
9.
Neural Dev ; 7: 26, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22776033

RESUMO

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Córtex Cerebral/fisiologia , Eletroporação/métodos , Técnicas de Transferência de Genes , Células-Tronco Neurais/citologia , Neurônios/fisiologia , Pia-Máter/fisiologia , Animais , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Camundongos , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Neuroglia/citologia , Neuroglia/fisiologia , Neurônios/citologia , Pia-Máter/citologia
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