AbstractHere we report the expansion of the genetic code of Mus musculus with various unnatural amino acids including Nɛ-acetyl-lysine. Stable integration of transgenes encoding an engineered Nɛ-acetyl-lysyl-tRNA synthetase (AcKRS)/tRNAPyl pair into the mouse genome enables site-specific incorporation of unnatural amino acids into a target protein in response to the amber codon. We demonstrate temporal and spatial control of protein acetylation in various organs of the transgenic mouse using a recombinant green fluorescent protein (GFPuv) as a model protein. This strategy will provide a powerful tool for systematic in vivo study of cellular proteins in the most commonly used mammalian model organism for human physiology and disease.