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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.
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
3.
J Forensic Sci ; 68(3): 839-855, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37078656

RESUMO

Recently, digital forensics has become increasingly important as it is used by investigation agencies, corporate, and private sector. To supplement the limitations of evidence capacity and be recognized in court, it is essential to establish an environment that ensures the integrity of the entire process ranging from collecting and analyzing to submitting digital evidence to court. In this study, common elements were extracted by comparing and analyzing ISO/IEC 17025, 27001 standards and Interpol and Council of Europe (CoE) guidelines to derive the necessary components for building a digital forensic laboratory. Subsequently, based on 21 digital forensic experts in the field, Delphi survey and verifications were conducted in three rounds. As a result, 40 components from seven areas were derived. The research results are based on the establishment, operation, management, and authentication of a digital forensics laboratory suitable for the domestic environment, with added credibility through collection of the opinions of 21 experts in the field of digital forensics in Korea. This study can be referred to in establishing digital forensic laboratories in national, public, and private digital forensic organizations as well as for employing as competency measurement criteria in courts to evaluate the reliability of the analysis results.


Assuntos
Ciências Forenses , Laboratórios , Ciências Forenses/métodos , Reprodutibilidade dos Testes , Controle de Qualidade , Medicina Legal
4.
Opt Express ; 19 Suppl 5: A1135-40, 2011 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-21935256

RESUMO

We investigate the effects of the refractive index of the encapsulant on the light-extraction efficiency (LEE) of light-emitting diodes (LEDs) for GaN LEDs (n ≈ 2.5) and AlGaInP LEDs (n ≈ 3.0). For non-absorbing rectangular parallelepiped LED chips, as the refractive index of the encapsulant increases, the LEE first increases quasi-linearly, then increases sub-linearly, and finally a saturation is reached. Furthermore, LEDs with a dual-layer graded-refractive-index (GRIN) encapsulant (n(encapsulant 1) = 1.57 and n(encapsulant 2) = 1.41) is fabricated through a two-step curing process. We demonstrate that such an LED further enhances the LEE by reducing Fresnel reflection loss at the encapsulant/air interface by 35% compared with an LED encapsulated with a single-layer encapsulant (n(encapsulant) = 1.57).

5.
Neurosci Res ; 143: 44-52, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29857015

RESUMO

We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages-P0, P7, and P14-when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ∼7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.


Assuntos
Eletroporação/métodos , Biossíntese de Proteínas , Células Piramidais/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Proteína Duplacortina , Desenvolvimento Embrionário , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células Piramidais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Ribossômica L10 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapsinas/metabolismo
6.
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
7.
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
8.
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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