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1.
PLoS Pathog ; 6(5): e1000910, 2010 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-20502631

RESUMEN

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.


Asunto(s)
Técnicas de Cultivo de Célula , Hepacivirus/crecimiento & desarrollo , Hepacivirus/genética , Hepatitis C/virología , Transfección/métodos , Adenoviridae/genética , Animales , Antivirales/farmacología , Muerte Celular , Chlorocebus aethiops , Genotipo , Hepacivirus/inmunología , Hepatitis C/sangre , Hepatitis C/tratamiento farmacológico , Anticuerpos contra la Hepatitis C/farmacología , Antígenos de la Hepatitis C/genética , Humanos , Interferón-alfa/genética , Interferón beta/genética , Pruebas de Neutralización , Estabilidad del ARN , ARN Viral/farmacología , Células Vero
2.
Viruses ; 14(7)2022 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-35891553

RESUMEN

Over the past two years, scientific research has moved at an unprecedented rate in response to the COVID-19 pandemic. The rapid development of effective vaccines and therapeutics would not have been possible without extensive background knowledge on coronaviruses developed over decades by researchers, including Kathryn (Kay) Holmes. Kay's research team discovered the first coronavirus receptors for mouse hepatitis virus and human coronavirus 229E and contributed a wealth of information on coronaviral spike glycoproteins and receptor interactions that are critical determinants of host and tissue specificity. She collaborated with several research laboratories to contribute knowledge in additional areas, including coronaviral pathogenesis, epidemiology, and evolution. Throughout her career, Kay was an extremely dedicated and thoughtful mentor to numerous graduate students and post-doctoral fellows. This article provides a review of her contributions to the coronavirus field and her exemplary mentoring.


Asunto(s)
Coronavirus Humano 229E , Receptores de Coronavirus , Animales , COVID-19 , Historia del Siglo XXI , Humanos , Ratones , Pandemias , Glicoproteína de la Espiga del Coronavirus/genética
3.
J Neurochem ; 84(4): 689-97, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12562513

RESUMEN

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.


Asunto(s)
Encefalinas/genética , Expresión Génica/fisiología , Integrasas , Precursores de Proteínas/genética , Elementos de Respuesta/fisiología , Proteínas Virales , Región de Flanqueo 5'/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Secuencia Conservada , Encefalinas/biosíntesis , Femenino , Marcación de Gen , Genes Reporteros , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , ARN Mensajero/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Elementos de Respuesta/genética , Células Madre/citología
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