Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system

AUTOR(ES)

Voon, Dominic C.

FONTE

Oxford University Press

RESUMO

Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose–response titrations.

Documentos Relacionados

• Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.
• The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA.
• Identification of a Novel Dexamethasone-Sensitive RNA-Destabilizing Region on Rat Monocyte Chemoattractant Protein 1 mRNA
• Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA.
• Highly stable and efficient mRNA templates for mRNA–protein fusions and C-terminally labeled proteins