ArticleNucleic acids research2002
Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery.
Article in Nucleic acids research, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 23 citations in OpenAlex.
- Incorporation of nonstandard amino acids into proteins: principles and applications.World journal of microbiology & biotechnology · 2020Review
- Stress Response and Adaptation Mediated by Amino Acid Misincorporation during Protein Synthesis.Advances in nutrition (Bethesda, Md.) · 2016Review
- Mistranslation of the genetic code.FEBS letters · 2014Review
- Electrical detection of cellular penetration during microinjection with carbon nanopipettes.Nanotechnology · 2014Article
- Sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major.Eukaryotic cell · 2007Article
- Reversible site-selective labeling of membrane proteins in live cells.Nature biotechnology · 2004Article
- Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.Nucleic acids research · 2004Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.
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