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. 2002 Dec 1;30(23):e128.
doi: 10.1093/nar/gnf128.

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

Erwin Ilegems  1 , Horst M Pick, Horst Vogel
Affiliations

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

Erwin Ilegems et al. Nucleic Acids Res. .

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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Figures

Figure 1
Figure 1
Scheme of the cloning steps for the wild-type (pT7PEGFP) and mutant EGFP (pT7PEGFPam64L) encoding plasmids used for the in vitro transcription. PCR amplification with primers 1 and 2 was performed to add a T7 promoter to the 5′ end and a poly(A) tail to the 3′ end of the EGFP coding sequence. Primers 3 and 4 were used for replacing the leucine 64 codon by TAG, and providing silent mutations for clonal selection. All nucleotide sequence modifications are shown in bold letters.
Figure 2
Figure 2
Scheme for the in vitro synthesis of the suppressor tRNA from a synthetic DNA template by T7 run-off transcription. Two synthetic complementary oligonucleotides encoding the E.coli-derived suppressor tRNAAlaCUA were annealed and cloned into the pCR4 vector, leading to pEcoliAlaCUA. T7 RNA polymerase run-off transcription was performed on the purified EcoRI and BsaI fragment giving rise to a 76mer suppressor tRNA.
Figure 3
Figure 3
In vivo suppression visualized by EGFP fluorescence recovery. (A) Schematic view of the assay for the amber suppressor tRNA function: co-injection of a mutated EGFP mRNA containing an amber codon at position 64 with an amber suppressor leucyl-tRNA. A successful suppression leads to complete translation of the mRNA transcript, thus to the appearance of the EGFP molecule. (B) Laser-scanning confocal micrograph of CHO cells visualizing EGFP fluorescence 20 h after co-injection of the mutated EGFP-mRNA with the amber suppressor tRNA. Size bar is 10 µm.
Figure 4
Figure 4
Correlation between injection volume and number of EGFP molecules per cell by microinjection of variable volumes of a solution containing 500 ng/µl rEGFP and 250 ng/µl Alexa546 in CHO cells. The injected volume is calculated on the basis of the known cell volume (see text for detailed description) and the final concentration of Alexa546 dye measured directly in the cell (based on a calibration curve established before).
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