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 glutaminyl-tRNA 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.

ACKNOWLEDGMENTS

We thank Herman Bujard for plasmids pUHD10-3 and the pUHD15-1, Phil Sharp and Leslie Leinwand for comments and suggestions on the manuscript, and Harold Drabkin for suggestions and encouragement. We also thank Annmarie McInnis for her usual patience, care, and cheerfulness in the preparation of the manuscript.

This work was supported by grants GM46942 and GM17151 from the National Institutes of Health and by a postdoctoral fellowship from the Human Frontier Science Program (to Ho-Jin Park).