A cell-based high-throughput screening assay for Farnesoid X receptor agonists / 生物医学与环境科学(英文)
Biomedical and Environmental Sciences
; (12): 465-469, 2007.
Article
en En
| WPRIM
| ID: wpr-249824
Biblioteca responsable:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.</p><p><b>METHODS</b>cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.</p><p><b>RESULTS</b>After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.</p><p><b>CONCLUSION</b>A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.</p>
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WPRIM
Asunto principal:
Plásmidos
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Factores de Transcripción
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Secuencia de Bases
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Transfección
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Línea Celular
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Química
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Reproducibilidad de los Resultados
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Receptores Citoplasmáticos y Nucleares
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ADN Complementario
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Cartilla de ADN
Tipo de estudio:
Diagnostic_studies
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Prognostic_studies
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Screening_studies
Límite:
Humans
Idioma:
En
Revista:
Biomedical and Environmental Sciences
Año:
2007
Tipo del documento:
Article