title: Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system.
abstract: 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.
authors.affiliation: GeneStream Pty Ltd 96 Chipping Road, City Beach, WA 6015, Australia.
authors.forename: Dominic C
authors.initials: DC
authors.lastname: Voon
authors.forename: Lily S
authors.initials: LS
authors.lastname: Subrata
authors.forename: Svetlana
authors.initials: S
authors.lastname: Baltic
authors.forename: Marco P
authors.initials: MP
authors.lastname: Leu
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Whiteway
authors.forename: Agnes
authors.initials: A
authors.lastname: Wong
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Knight
authors.forename: Frank T
authors.initials: FT
authors.lastname: Christiansen
authors.forename: John M
authors.initials: JM
authors.lastname: Daly
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D005822:Genetic Vectors; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D011506:Proteins / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D038621:Regulatory Sequences, Ribonucleic Acid; D009626:Terminology as Topic; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid
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