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, 133 (36), 14196-9

Expanding the Genetic Code of an Animal

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Expanding the Genetic Code of an Animal

Sebastian Greiss et al. J Am Chem Soc.

Abstract

Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode Caenorhabditis elegans.

Figures

Figure 1
Figure 1
Expanding the genetic code of C. elegans. (A) DNA constructs used for genetic code expansion in C. elegans. (B) Amino acid substrates for PylRS used in this study.
Figure 2
Figure 2
Each of the components required for genetic code expansion in C. elegans is expressed in the animal. (A) Effect of nonsense-mediated decay (NMD) on GFP expression levels from worms containing the reporter construct Prps-0::mGFP-TAG-mCherry-HA-NLS. “Wild-type C. elegans” shows the GFP fluorescence of a representative wild-type animal. “ΔNMD” shows the GFP fluorescence of a transgenic worm created by crossing the reporter construct into the smg-2(e2008) mutant background. (B) FLAG-MmPylRS (top) and MmtRNACUA (bottom) are expressed from animals containing Prps-0::FLAG-MmPylRS and PCeN74-1::MmPylT. FLAG-MmPylRS was detected by western blot in worm lysates using an anti-FLAG antibody. MmtRNACUA was detected by northern blot from total RNA isolated from worms. All experiments used a mixed-stage population.
Figure 3
Figure 3
The orthogonal MmPylRS/MmtRNACUA pair incorporates 1 or 2 in response to the amber codon in C. elegans. (A) Fluorescence images of worms containing Ex1 [Prps-0::mGFP-TAG-mcherry-HA-NLS, Prps-0::FLAG-MmPylRS, PCeN74-1::MmPylT, Prps-0::hpt] in the absence (top) and in the presence (bottom) of 1 (see also SI Movies 1–4). Both GFP and the GFP::mCherry fusion protein show mosaic expression throughout the animal. GFP mosaicism is not visible here because its fluorescence is saturating under the imaging conditions, but GFP mosaicism is clear in Figure 2A. mCherry is nuclear localized, making its mosaicism more obvious. (B) Lanes 1–6: western blot of lysates of worms grown in the absence or presence of 1. Lanes 7–9: western blot of GFP::mCherry fusion protein affinity purified using an antibody against mCherry. GFP::mCherry was detected using an antibody against the C-terminal HA tag. Anti-GFP blots are loading controls (lanes 1–6) and input controls (lanes 7 and 8). More protein was loaded in the “no added amino acid” lanes (lanes 1 and 4). (C) Genetically encoded 2 can be specifically labeled with an azido derivative of biotin, 3. Immunoprecipitated GFP::mCherry fusion from B was labeled with 3 and the reaction products were detected using streptavidin.

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