Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors.
Assay Drug Dev Technol
; 1(6): 777-87, 2003 Dec.
Article
en En
| MEDLINE
| ID: mdl-15090224
ABSTRACT
Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.
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Banco de datos:
MEDLINE
Asunto principal:
Beta-Lactamasas
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Receptores Citoplasmáticos y Nucleares
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Transferencia Resonante de Energía de Fluorescencia
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Hígado
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Miniaturización
Límite:
Animals
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Humans
Idioma:
En
Año:
2003
Tipo del documento:
Article