Assay for investigating nonsense mutations
Highthroughput assay system for rapid whole mammalian cell analysis which provides information about the impact of nonsense mutations on protein translation and identify factors that modulate translation readthrough.
Development of the single cell-based translation readthrough assay. (A) An outline of the assay. In the event of translation termination at the internal PTC, a DsRed-Express protein will be generated whilst readthrough will produce a fusion dual-fluorescence protein (DsRed Express and GFP). HEK293 cells transfected with wild- type construct generated dual-fluorescence (Bi) whilst construct containing internal PTC produced single fluorescence (Bii), which generated dual-fluorescence cells following G418 sulfate treatment (Biii).
Nonsense mutations occur in between 15% to 30% of all inherited diseases such as cystic fibrosis, haemophilia, retinitis pigmentosa and Duchenne muscular dystrophy, and in 5-10% of the major genetic diseases including many forms of cancer, for example, colorectal cancer. A nonsense mutation changes a codon that originally specified an amino acid into a stop codon which will cause premature termination of mRNA translation (and a truncated protein to be produced) or may cause the mRNA to become destabilised and degraded by the nonsense mediated decay (NMD) pathway. Both events are likely to reduce the abundance of the full-length active protein.
The investigation of the functional consequences of nonsense mutations and any subsequent development of therapies therefore relies on understanding whether mutation triggers the NMD process or results in abnormal protein synthesis. Drugs which promote readthrough of disease-causing premature termination codons might alleviate the effects of nonsense mediated diseases. Currently, there are no high throughput screening assays which allow the consequences of nonsense mutations to be determined.
King’s has developed a dual-florescence reporter assay whereby the effect of nonsense mutations on protein translation can be visualised in single live mammalian cells in a high throuput format.
Functional analysis of consequences of nonsense mutation of protein production.
Screening assay for identifying agents which promote readthrough of disease-causing premature termination codons.
Assay provides for an instant and comprehensive insight into whether this genetic variation leads to either abnormal protein synthesis or triggering of the NMD pathway.
Assay can be implemented in to high throughput formats and can thus be used for rapid screening of agents, such as cDNA, chemical and peptide libraries, to identify those factors that promote or inhibit protein translation and NMD.
Nonsense readthrough and splicing assays (filed on 12 March 2008; Application No. US 61/035,855). Applicant: King’s College London, UK. Inventors: Professor Richard Trembath & Dr Talat Nasim.
This technology is available as an Easy Access licence deal to companies and individuals.
For more information on this technology, please contact:
Michael Jorgensen PhD
IP & Licensing Manager
King's College London
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