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2.
Assay Drug Dev Technol ; 4(3): 253-62, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16834531

RESUMO

Retina-specific nuclear receptor (RNR), also known as PNR and NR2E3, is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. Here we describe homogeneous cell-based resonance energy transfer assay for identification of RNR agonists using beta-lactamase as the reporter gene. Bacterial beta-lactamase reporter construct containing GAL4 response elements was randomly integrated into the genome with subsequent selection of responsive cell pools by fluorescence-activated cell sorting. Chimeric RNR (RNR hinge and ligand-binding domains fused to GAL4 DNA-binding domain) was stably transfected into mammalian Flp-In Chinese hamster ovary cells using Flp-mediated recombination into a single pre-integrated Flp recombination target site. Since no RNR ligand could be used as a control for monitoring the development of the RNR assay, we developed a parallel cell line with the functionally related well-characterized thyroid hormone nuclear receptor. This parallel thyroid hormone nuclear receptor system was used as a "guide" in optimizing the RNR assay for ultra-high-throughput screening in 3,456-well nanoplate format. The assay was successfully used to screen a large compound collection for RNR agonists. In this study we demonstrated the feasibility of developing and optimization of the high-throughput screening-compatible assay for the orphan nuclear receptor in the absence of its cognitive ligand.


Assuntos
Bioensaio/métodos , Citometria de Fluxo/métodos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Receptores Nucleares Órfãos , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/antagonistas & inibidores
3.
Cytometry A ; 65(2): 133-9, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15825164

RESUMO

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Fluoresceínas/química , Genes Reporter , Lactamas/química , Espectrometria de Fluorescência/métodos , beta-Lactamases/genética , Animais , Células CHO , Calibragem , Cricetinae , Citometria de Fluxo/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Polyomavirus/genética , Poliestirenos/química , Regiões Promotoras Genéticas
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