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1.
Cell ; 171(6): 1437-1452.e17, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-29195078

RESUMO

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.


Assuntos
Perfilação da Expressão Gênica/métodos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/economia , Humanos , Neoplasias/tratamento farmacológico , Especificidade de Órgãos , Preparações Farmacêuticas/metabolismo , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/métodos , Bibliotecas de Moléculas Pequenas
2.
Nature ; 447(7145): 679-85, 2007 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-17554301

RESUMO

Until now, animal cloning and the production of embryonic stem cell lines by somatic cell nuclear transfer have relied on introducing nuclei into meiotic oocytes. In contrast, attempts at somatic cell nuclear transfer into fertilized interphase zygotes have failed. As a result, it has generally been assumed that unfertilized human oocytes will be required for the generation of tailored human embryonic stem cell lines from patients by somatic cell nuclear transfer. Here we report, however, that, unlike interphase zygotes, mouse zygotes temporarily arrested in mitosis can support somatic cell reprogramming, the production of embryonic stem cell lines and the full-term development of cloned animals. Thus, human zygotes and perhaps human embryonic blastomeres may be useful supplements to human oocytes for the creation of patient-derived human embryonic stem cells.


Assuntos
Cromossomos de Mamíferos/genética , Células-Tronco Embrionárias/citologia , Mitose , Técnicas de Transferência Nuclear , Zigoto/citologia , Zigoto/metabolismo , Aneuploidia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Interfase , Masculino , Camundongos , Mitose/efeitos dos fármacos , Nocodazol/farmacologia , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento
3.
Nat Genet ; 53(1): 86-99, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33414553

RESUMO

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.


Assuntos
Variações do Número de Cópias de DNA/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento do Exoma
5.
Curr Biol ; 23(1): 21-31, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23177476

RESUMO

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.


Assuntos
Blastômeros/citologia , Desenvolvimento Embrionário , Animais , Blastômeros/fisiologia , Linhagem da Célula , Feminino , Proteínas Luminescentes/análise , Masculino , Camundongos , Camundongos Transgênicos , Recombinação Genética , Proteína Vermelha Fluorescente
6.
PLoS One ; 7(11): e49490, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166684

RESUMO

The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3' untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
7.
Proc Natl Acad Sci U S A ; 101(1): 233-8, 2004 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-14679297

RESUMO

Virus infection, double-stranded RNA, and lipopolysaccharide each induce the expression of genes encoding IFN-alpha and -beta and chemokines, such as RANTES (regulated on activation, normal T cell expressed and secreted) and IP-10 (IFN-gamma inducible protein 10). This induction requires the coordinate activation of several transcription factors, including IFN-regulatory factor 3 (IRF3). The signaling pathways leading to IRF3 activation are triggered by the binding of pathogen-specific products to Toll-like receptors and culminate in the phosphorylation of specific serine residues in the C terminus of IRF3. Recent studies of human cell lines in culture have implicated two noncanonical IkappaB kinase (IKK)-related kinases, IKK-epsilon and Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1), in the phosphorylation of IRF3. Here, we show that purified recombinant IKK-epsilon and TBK1 directly phosphorylate the critical serine residues in IRF3. We have also examined the expression of IRF3-dependent genes in mouse embryonic fibroblasts (MEFs) derived from Tbk1(-/-) mice, and we show that TBK1 is required for the activation and nuclear translocation of IRF3 in these cells. Moreover, Tbk1(-/-) MEFs show marked defects in IFN-alpha and -beta, IP-10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the Toll-like receptors 3 and 4 by double-stranded RNA and lipopolysaccharide, respectively. Finally, TRIF (TIR domain-containing adapter-inducing IFN-beta), fails to activate IRF3-dependent genes in Tbk1(-/-) MEFs. We conclude that TBK1 is essential for IRF3-dependent antiviral gene expression.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Serina-Treonina Quinases/deficiência , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Expressão Gênica , Quinase I-kappa B , Técnicas In Vitro , Fator Regulador 3 de Interferon , Interferon beta/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo
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