Generation of Stable Amber Suppression Cell Lines

Elsässer SJ

Methods in molecular biology (Clifton, N.J.) 1728 (-) 237-245 [2018-00-00; online 2018-02-06]

Noncanonical amino acid mutagenesis via amber suppression provides the means to tailor proteins inside living cells. A wide range of noncanonical amino acids have been incorporated using the Methanococcus pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/PylT) in mammalian cell systems in proof of principle experiments, for (1) minimal genetically encoded fluorescence or affinity tagging, (2) photo-control of enzymes, (3) genetically encoded posttranslational protein modifications. We have developed a general and efficient method to genomically integrate the PylRS/PylT amber suppression machinery using PiggyBac-mediated transposition. A general protocol for the generation of stable amber suppression cell lines is described here. Using the modular plasmid system, homogenous and highly efficient amber suppression in a wide range of cell lines can be achieved.

SciLifeLab Fellow

Simon Elsässer

PubMed 29405002

DOI 10.1007/978-1-4939-7574-7_15

Crossref 10.1007/978-1-4939-7574-7_15

Publications 9.5.0