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. 2023 Apr 14;9(15):eadf2695. doi: 10.1126/sciadv.adf2695

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Fig. 2. — (A) The incorporation of LSL-DdCBE into rat Rosa26 locus using CRISPR-Cas9–mediated homologous recombination. (B and C) Genotyping of LSL-DdCBE-Atp8 KI (B) and LSL-DdCBE-Nd1 KI (C) F₀ rats by PCR with upstream primer pair (Up band), downstream primer pair (Down band), and internal primer pair (DddA_tox band). (D) The mtProteins KO in the heart tissue induced by crossing LSL-DdCBE KI rat with α-MHC-Cre rat. (E and F) The DdCBE expression in the heart tissue of Atp8^MHC-Cre (E) and Nd1^MHC-Cre (F) rat was detected by Western blot using the anti-Flag antibody. (G) The localization of DdCBE in mitochondrial of Atp8^MHC-Cre and Nd1^MHC-Cre heart tissues. The anti-Flag (green) for DdCBE, anti-TOMM20 for mitochondria (red), and 4′,6-diamidino-2-phenylindole (DAPI) for nucleus (blue). Scale bars, 10 μm. (H) The frequency of DdCBE-mediated C·G-to-T·A conversion at Atp8 G7783 and Nd1 G2996 sites in the heart, liver, spleen, kidney, muscle, brain, and toe of Atp8^MHC-Cre and Nd1^MHC-Cre rats was analyzed by deep sequencing. (I and J) Protein level of ATP8 (I) and ND1 (J) in tissues of Atp8^MHC-Cre and Nd1^MHC-Cre was detected by Western blot, respectively.