High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.
Methods Mol Biol
; 1755: 75-87, 2018.
Article
em En
| MEDLINE
| ID: mdl-29671264
ABSTRACT
Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.
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Base de dados:
MEDLINE
Assunto principal:
Cromossomo X
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Genes Reporter
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Interferência de RNA
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Inativação do Cromossomo X
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Luciferases
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article