Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Ilegems E,Pick HM,Vogel H

doi

10.1093/nar/gnf128

keywords:

subject

Has Abstract

pub_date

2002-12-01 00:00:00

pages

e128

issue

23

eissn

0305-1048

issn

1362-4962

journal_volume

30

pub_type

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