Figure 2. Mutant human G11778A ND4 transmission in mice. A: RT–PCR of RNA extracted from retinal mitochondria (RM), optic nerve mitochondria (OM), retinal nuclear (RN), or optic nerve nuclear (ON) debris, retinal cytoplasm (RC), and optic nerve cytoplasmic (OC) fractions of experimental right eyes had the expected 500 bp band for ND4FLAG that was absent in RNA extracted from control left eyes infected with scAAV-GFP. B: A sequencing chromatograph shows the corresponding DNA sequence to be that of the mutant human ND4 where the base adenine (A; arrows) has replaced guanine (G). C: One of the sequences, SEQmutND41-OM, is aligned to the wild-type human ND4 (ND4mito) showing this G to A transition (arrows). It also reveals the sequence of the mouse ND4 (mitoND4mouse) confirmed that the PCR products were indeed mutant human G11778A ND4, further supporting that exogenous ND4 was imported into retinal and optic nerve mitochondria by a mitochondria-targeted AAV where it was transcribed. D: Immunoblotting of isolated optic nerve and retinal mitochondria showed that the MTS-targeted AAV directed the synthesis of mutant human ND4FLAG in the experimental eyes, but the control eyes injected with GFP were negative for FLAG. ND4 was overexpressed in experimental eyes relative to the endogenous ND4 of GFP injected control eyes. Expression of NDUFS4, a nuclear encoded complex I subunit, is shown for housekeeping. E: Amino acid sequence of ND4 with the start methionine (met) shows that the TGA codon is a termination sequence for protein synthesis in the cytoplasm, but specifies the amino acid tryptophan for synthesis within the mitochondrial ribosomes. F: Illustrating that full-length ND4 with 340 amino acids can be expressed only within mitochondria.