Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system.
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.
Voon
Dominic C
DC
GeneStream Pty Ltd 96 Chipping Road, City Beach, WA 6015, Australia.
Subrata
Lily S
LS
Baltic
Svetlana
S
Leu
Marco P
MP
Whiteway
Joanna M
JM
Wong
Agnes
A
Knight
Samuel A
SA
Christiansen
Frank T
FT
Daly
John M
JM
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
D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid
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