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. 2024 Jan 30;11(13):2306792. doi: 10.1002/advs.202306792

Figure 5.

Figure 5

Efficiency of NAEK incorporation evaluated apparently in the heart, brain, muscle, and stomach tissues of transgenic mice. A) Illustrates the strategic methodology employed for engineering three distinct types of transgenic mice, each harboring an orthogonal aaRS/tRNA pair gene, laying the groundwork for specific UAA incorporation. B) Presents a comparative analysis of pylRS and tRNApyl transcription levels between the transgenic and WT mice, offering quantitative insights into the genetic modification's effectiveness (n = 3). Data are expressed as mean ± S.D., with statistical significance determined by an unpaired two‐tailed Student's t‐test (**** p < 0.0001). C) Depicts PCR analysis of tail DNA from successive generations of the transgenic mice, utilizing two specific primers (Primer 1 targeting a sequence at 900 bp and Primer 2 targeting a sequence at 300 bp) to confirm the presence and inheritance of the modified genes (n = 3), thereby verifying the successful establishment of the transgenic lines. D) Demonstrates the efficacy of NAEK incorporation through Western blot analysis of GFP restoration in transgenic mice post one‐week daily intramuscular injections of ChNAEK (20 and 30 mg) and NAEK aqueous solution (30 mg) (n = 2). E) Evaluates GFP restoration in the heart, brain, muscle, and stomach tissues of PylRS‐tRNA‐GFP39TAA transgenic mice via Western blot, following 1 week of oral administration of equivalent molar doses of ChNAEK or NAEK solution (30 mg) (n = 2). F) Provides Western blot analysis of GFP restoration in transgenic mice post 1‐week oral administration of various doses (10, 30, and 50 mg) of ChNAEK (n = 2), illustrating the dose‐dependent efficacy of the oral formulation. The integrated optical density (IOD) of GFP bands from panels D, E, and F, normalized to wild‐type GFP levels and corrected for sample loading, quantitatively underscores the superior restoration capabilities of ChNAEK. Normalized IOD values are provided beneath each Western blot. These comprehensive analyses collectively confirm the enhanced efficiency of NAEK incorporation in vital organs of transgenic mice through the UAA‐based IL formulation, highlighting its potential in precision therapeutic applications.