RESUMO
Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Engenharia Genética , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Eletroporação , Genoma , Dados de Sequência Molecular , Fator de Transcrição PAX7/fisiologiaRESUMO
Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.
Assuntos
Coturnix/genética , Técnicas de Transferência de Genes , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Coturnix/crescimento & desenvolvimento , Feminino , Masculino , Plasmídeos/genética , Transfecção , NavegadorRESUMO
The fusion of myoblasts into multinucleated myotubes is a crucial step of muscle growth during development and of muscle repair in the adult. While multiple genes were shown to play a role in this process, a vertebrate model where novel candidates can be tested and analyzed at high throughput and relative ease has been lacking. Here, we show that the early chicken embryo is a fast and robust model in which functional testing of muscle fusion candidate genes can be performed. We have used known modulators of muscle fusion, Rac1 and Cdc42, along with the in vivo electroporation of integrated, inducible vectors, to show that the chicken embryo is a suitable model in which their function can be tested and quantified. In addition to nuclei content, specific characteristics of the experimental model allow a fine characterization of additional morphological features that are nearly impossible to assess in other model organisms. This study should establish the chicken embryo as a cheap, reliable and powerful model in which novel vertebrate muscle fusion candidates can be evaluated.
Assuntos
Desenvolvimento Muscular , Mioblastos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Núcleo Celular/metabolismo , Embrião de Galinha , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mioblastos/citologia , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Genome editing is driving a revolution in the biomedical sciences that carries the promise for future treatments of genetic diseases. The CRISPR/Cas9 system of RNA-guided genome editing has been successfully applied to modify the genome of a wide spectrum of organisms. We recently showed that this technique can be combined with in vivo electroporation to inhibit the function of genes of interest in somatic cells of the developing chicken embryo. We present here a simplified version of the previously described technique that leads to effective gene loss-of-function.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Eletroporação , Fibroblastos/citologia , Fibroblastos/metabolismo , Genoma , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
BACKGROUND: PAPRICA is a research program designed to estimate the impact on the health of patients with chronic respiratory insufficiency of a prevention strategy based on notification of ozone pollution. The first year of this study was conducted during the 2003 heat wave, and high temperatures were therefore considered as a confounding factor in the data analysis. The aim of the present study was to assess the relationship between ozone and temperature in order to propose a methodology to distinguish between the effects of ozone and temperature on the impact of a prevention strategy with regard to ozone pollution. METHODS: Multivariate analyses were used to identify associated climate and ozone pollution profiles. This descriptive method is of great value to highlight the complexity of interactions between these parameters. RESULTS: Ozone concentration and temperature were strongly correlated, but the health impact of ozone pollution alone will be evaluated by focusing on situations characterized by ozone concentrations above 110 mug/m3/8h (air quality guidelines to protect human health defined by the French legislation) and temperatures lower than 26 degrees C, below the discomfort threshold. CONCLUSION: The precise relationship between ambient ozone concentration and temperature identified during the PAPRICA 2003 study period will be used in analysing the PAPRICA health data.
Assuntos
Poluição do Ar/análise , Temperatura Alta/efeitos adversos , Disseminação de Informação , Ozônio/análise , Insuficiência Respiratória/complicações , Poluição do Ar/efeitos adversos , Poluição do Ar/prevenção & controle , Atmosfera/análise , Atmosfera/química , Doença Crônica , Fatores de Confusão Epidemiológicos , Monitoramento Ambiental , França , Humanos , Concentração Máxima Permitida , Análise Multivariada , Ozônio/toxicidade , Estações do Ano , Perfil de Impacto da DoençaRESUMO
Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.
Assuntos
Movimento Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Animais , Autoantígenos/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Citocinas/biossíntese , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Interferon gama/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Esclerose Múltipla Recidivante-Remitente/terapia , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Solubilidade , Baço/citologia , Baço/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Resultado do Tratamento , Cordão Umbilical/citologiaRESUMO
Gliomas are the most common malignant primary brain tumors in adults. The median survival never exceeds 12 months, owing to inherent resistance to both radio and chemotherapies. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in glioblastomas (GBM), making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. Inhibition of cell proliferation and induction of apoptosis by erlotinib were investigated in U87-MG and DBTRG-05MG, two human glioblastoma cell lines. The expression of several apoptosis-related proteins was investigated in these cell lines and in tumoral tissue from glioblastomas. Both cell lines expressed wild-type EGFR but were deficient for PTEN. Erlotinib induced a marked accumulation of the BIM protein, but the activation of caspase-3 machinery was missing, regardless of the decrease in XIAP. Moreover, in U87-MG, erlotinib promoted accumulation of αB-crystallin a small heat shock protein capable to impair caspase activation. DBTRG-05MG was found deficient for procaspase 3 and constitutively overexpressed αB-crystallin. Similarly, deficiencies in PTEN and procaspase 3 were constantly found in samples from glioblastoma samples, while αB-crystallin expression was inconsistent. In cell lines, high concentrations of erlotinib induced cell death through a caspase independent process and an autophagic process was evidenced in U87-MG. Inhibition of autophagy induced a marked increase in the death-inducing activity of erlotinib. These results confirm that glioblastoma cell lines exhibit several anti-apoptotic mechanisms, and underline that EGFR targeted therapy must be associated to other inhibitors to achieve an antitumoral effect.