Fig 2: Generation and characterization of CNT1-deficient mice.A Schematic workflow illustrating the Slc28a1 editing and genotyping strategy. B Representative DNA chromatogram illustrating the sequence alignment between the Slc28a1 reference and experimental sequence from Slc28a1+/+ and Slc28a1−/− mice with deleted nucleic acids indicated with a dash (-). Sequencing performed in two independent experiments. C Representative genotyping gel loaded with PCR-amplified Slc28a1 from Slc28a1+/+ and Slc28a1−/− with and without the Acul restriction digest. Genotyping performed in over ten independent experiments. D Relative Slc28a1 gene expression in Slc28a1−/− mouse organs normalized to GAPDH and Slc28a1+/+ control displayed as a fold change (2−ΔΔCt). Data represent mean ± SEM (n = 3 mice/group mean ± SEM, *p < 0.05 by two-tailed t-test). Gene expression performed in two independent experiments. E Representative immunoblots of mCNT1 in Slc28a1+/+ and Slc28a1−/− mouse organs at 8 weeks of age with GAPDH as a loading control. Empty vector and mCNT1 overexpression (OE) controls were generated in HEK293 cells (#CRL 1573, ATCC). Immunoblotting performed in two independent experiments.