Figure 2. Lack of homologous recombination. A: An illustration of the mouse mitochondrial DNA shows that murine ND4 is flanked by two genes. B: An illustration of the mouse mitochondrial DNA shows that the adjacent gene encoding for ND4L (arrow) is at the 5′ end of murine ND4 (arrow). C: An illustration of the mouse mitochondrial genome shows that the gene for the tRNA histidine (arrow) is at the 3′ end of mouse ND4 (arrow). D: Alignment of 5′ human ND4 reads for mouse human chimeras shows only AAV vector sequences upstream of the start methionine codon (ATG) for ND4. If there were homologous recombination of human ND4 into the mouse mitochondrial genome replacing murine ND4, then nucleotides for mouse ND4L would be expected to be adjacent to the ATG methionine codon. E: Alignment of the 3′ human ND4 reads shows the FLAG epitope that we appended to human ND4 for immunodetection and adjacent vector sequences, but not the tRNA histidine that would have indicated homologous recombination. Sc-HSP-ND4=self-complementary AAV plasmid containing the heavy strand promoter (HSP) driving human ND4.