Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Abstract

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyltRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Rapid Mapping of Protein Structure, Interactions, and Ligand Binding by Misincorporation Proton-Alkyl Exchange

“…Alkylation of misincorporated cysteines in vivo would probe protein interactions in their natural environment. Presumably, the technique can be adapted to eukaryotic expression systems known to accommodate suppressor tRNAs (28,29). Taken together, these approaches will make possible detailed structural investigations of complex and formerly inaccessible biological processes.…”

Suppression of Nonsense Mutations in Cell Culture and Mice by Multimerized Suppressor tRNA Genes

“…These approaches induced repression or activation of constitutive suppressor tRNA expression (16,49,52) as well as control of suppressor tRNA function by modulation of the aminoacylation process (18,39).…”

Modulation of cell growth and transformation by doxycycline‐regulated FGF‐2 expression in NIH‐3T3 cells

“…On the basis of these observations, the establishment of a biological system that allowed the modulation of FGF-2 expression in mammalian cells would be useful (Seghezzi et al, 1998). The tetracycline-regulated gene expression system has been used to control the expression of heterologous genes in many cell types (Gossen et al, 1995;Howe et al, 1995;Park and Raj Bhandary, 1998;Wu and Chiang, 1996), as well as to alter the temporal expression of transgenes in mice (Chen et al, 1998;Furth et al, 1994;St. Onge et al, 1996).…”