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. 2017 Feb;27(2):294-297.
doi: 10.1038/cr.2016.145. Epub 2016 Dec 9.

Heritable expansion of the genetic code in mouse and zebrafish

Yuting Chen  1 , Ji Ma  2 , Wenqing Lu  1 , Meilin Tian  2   3 , Marion Thauvin  4 , Chonggang Yuan  2 , Michel Volovitch  4   5   6 , Qian Wang  7 , Jeff Holst  7 , Mingyao Liu  1 , Sophie Vriz  4   5   6   8 , Shixin Ye  3   5   6   9 , Lei Wang  10 , Dali Li  1
Affiliations

Heritable expansion of the genetic code in mouse and zebrafish

Yuting Chen et al. Cell Res. 2017 Feb.
No abstract available

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Figures

Figure 1
Figure 1
Generation and characterization of transgenic mice and zebrafish with an expanded genetic code. (A) Schematic diagram of the construct used to generate transgenic mice and the reporter construct. The transgene construct contains AzFRS and four copies (4×) of Bacillus stearothermophilus (Bst)-tRNACUA. pA, polyadenylation signal. The eGFP-TAG-mCherry reporter construct was generated by inserting an amber stop codon (TAG) between eGFP and mCherry genes. Verified in HEK293T and mouse embryonic fibroblast (MEF) cells, this reporter expresses mCherry only when tRNACUA, AzF-RS and AzF are all available to cells. (B) Western blot analysis of the indicated tissue samples from transgenic RS(tRNA)4× mice of F2 generation and descendants. Arrow indicates the immunoreactive band of AzFRS-FLAG detected by an anti-FLAG antibody. GAPDH serves as an internal loading control. (C) RNA-seq analysis of liver tissues isolated from 6-week-old WT and F2 transgenic RS(tRNA)4× mice. The plot shows whole-transcriptome fragments per kilobase of exon per million fragments mapped (FPKM). Significantly (P < 0.05; fold change > 2) up- and downregulated genes in the transgenetic mice from three biological replicates are colored red and blue, respectively; other genes are in black. (D-F) Genome-integrated orthogonal tRNACUA/AzFRS pair-directed AzF incorporation in response to the amber codon in primary cells isolated from transgenic mice of F2-F3 generation. Representative images are shown for primary neuronal cells (D), bone marrow cells (E) and fibroblasts (F) transduced with lentivirus carrying the reporter gene eGFP-amb-mCherry in the presence or absence of 1 mM AzF. Scale bar: 100 μm in D and F; 20 μm in E. (G) Schematic diagram of the construct used to generate transgenic zebrafish and the reporter construct. The transgene construct contains an orthogonal tRNACUA/AzFRS pair, SVEpA (SV40 early polyadenylation signal), SVLpA (SV40 late polyadenylation signal), γCRY (xenopus γ-crystallin promoter), U6 (human U6 pol III promoter), INS (SP-10 mouse insulator) and ubi (zebrafish ubiquitin promoter). (H) Western blot analysis of AzFRS-FLAG expression in embryos or adult caudal fin tissues in F2 generation using an anti-FLAG antibody (indicated by an arrow). The FLAG antibody also detects a non-specific band, migrating slower than the tagged AzFRS band. (I-K) Incorporation of AzF into the eGFP reporter in the transgenic zebrafish in vivo. 1-cell-stage embryos of F2-F3 generations of transgenic zebrafish were injected with the mRNA encoding the mCherry-eGFP (Y145amb) reporter. The embryos were incubated in water with or without 2.5 mM AzF for 24 h. Incorporation of AzF at the amber codon site allows full-length eGFP expression in the nucleus. The fluorescence images of whole embryos (I), and mesenchymal (J) and notochord (K) tissues are shown. Arrowheads indicate cells with AzF incorporation. Scale bars: 50 μm.
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References

    1. Wang L, Brock A, Herberich B, Schultz PG. Science 2001; 292:498–500. - PubMed
    1. Liu CC, Schultz PG. Annu Rev Biochem 2010; 79:413–444. - PubMed
    1. Parrish AR, She X, Xiang Z, et al. ACS Chem Biol 2012; 7:1292–1302. - PMC - PubMed
    1. Bianco A, Townsley FM, Greiss S, Lang K, Chin JW. Nat Chem Biol 2012; 8:748–750. - PubMed
    1. Kang JY, Kawaguchi D, Coin I, et al. Neuron 2013; 80:358–370. - PMC - PubMed

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